CN109804087A - Using quencher based on optical nano-pore sequencing - Google Patents

Abstract

The present invention relates to for using the method and composition based on the analysis of optical polynucleotides of nano-pore, wherein quencher is used to reduce or eliminate the stray optical signal that the polynucleotide labelling object outside by detection zone generates.In some embodiments, the present invention can be implemented by lower step: polynucleotides transposition being made to pass through nano-pore, wherein different types of nucleotide of polynucleotides generates the different fluorescent labels of differentiable fluorescence signal, and wherein nucleotide is limited to successively move and passes through detection zone by the nano-pore；Excite the fluorescent marker；Use the fluorescence signal of the fluorescent marker of excitation of one or more of non-fluorescent quencher quenching outside detection zone；When fluorescent marker passes through detection zone, the fluorescence signal emitted from fluorescent marker is detected；And the sequence of nucleotide is determined according to the fluorescence signal detected.

Description

Using quencher based on optical nano-pore sequencing

Cross reference to related applications

This application claims preferential in August in 2016 19 days the U.S. Provisional Patent Application submitted the 62/377,409th Power, content are hereby incorporated by reference in its entirety by reference.

Background of invention

The optical detection of nucleotide has been used as technical difficulties some in nano-pore sequencing field such as, such as simultaneously The difficult possibility solution for collecting independent signal from extensive nanohole array is suggested.It is deposited however, implementing optical means In many challenges, marked especially with respect to the unimolecule on polynucleotides of the apparent optical noise background measurement from transposition The difficulty of the optical signalling of object.Measurement from monomolecular fluorescence signal carries out in several cases, such as Lackowitz, Principles of Fluorescence Spectroscopy, the third edition (Springer, 2006)； Moerner et al., Review of Scientific Instruments, 74 (8): 3597-3619 (2003)；Michalet etc. People Ann.Rev.Biophys.Biomol.Struct., 32:161-182 (2003)；Including with based on stream (flow-based) Those of high speed DNA sequencing correlation situation, such as Jett et al., J.Biomol.Struct.Dyn., 7 (2): 301-309 (1989).However, new challenge is presented based on optical nano-pore sequencing, because it needs to measure from quickly through nanoscale The continuous optical signal of marker on the complete polynucleotide of detection zone.Particularly, the optical markings object outside detection zone may There is notable contribution to the noise in the signal of collection.

In view of the above circumstances, if method can be used for eliminating or reducing the optical noise of the marker outside detection zone, It will be improved based on optical nano-pore sequencing.

Summary of the invention

The present invention relates to for using the method and composition based on the analysis of optical polynucleotides of nano-pore.At one Aspect, the present invention relates to the stray optical signals that quencher reduces or eliminates the generation of the polynucleotide labelling object outside by detection zone The purposes of (spurious optical signals).

In one aspect, it the present invention relates to a kind of method of analysis of polynucleotide, the described method comprises the following steps: (a) Make polynucleotides transposition by nano-pore, wherein different types of nucleotide of the polynucleotides generates differentiable fluorescence The different fluorescent labels of signal, and wherein nucleotide is limited to successively move and passes through detection zone by nano-pore；(b) swash Send out fluorescent marker described；(c) fluorescence of the fluorescent marker of the excitation using non-fluorescent quencher quenching outside detection zone Signal；(d) when fluorescent marker passes through detection zone, the fluorescence signal emitted from the fluorescent marker is detected；And (e) root The sequence of nucleotide is determined according to the fluorescence signal detected.

On the other hand, the present invention relates to a kind of method of the nucleotide sequence of determining polynucleotides, the method packets It includes following steps: nanohole array (a) is provided, the nano-pore battle array sequence includes with the first side, second side and to pass through More than one (a plurality of) nano-pore immobilon-p, the immobilon-p separates the first Room and second Room, so that often A nano-pore provides the fluid communication between the first Room and second Room, and each nano-pore has and leads to the outlet of second Room simultaneously And size is customized so that single-chain nucleic acid can not pass through by double-strandednucleic acid, and each nano-pore is by the polynucleotides of transposition Nucleotide be limited to successively move the detection zone by its exit；(b) polynucleotides is made to pass through nano-pore from the first Room Transposition is to second Room, and each polynucleotides have one or more fluorescent markers for being attached to its nucleotide, so that not Congener nucleotide generates the different fluorescent labels of differentiable fluorescence signal；(c) it is irradiated with excitation beam glimmering Signal object；(d) fluorescent marker of the excitation outside detection zone is quenched with the non-fluorescent quencher in conjunction with polynucleotides Fluorescence signal；(e) fluorescence signal of fluorescent marker of the detection in detection zone；(f) according to the fluorescence signal detected Determine the nucleotide sequence of polynucleotides.

The present invention suppresses or eliminates the fluorescence signal of the generation of the fluorescent marker outside by detection zone by providing quencher The undesired optical noise of form advantageously overcomes the problem above based on optical nano-pore sequencing field.The present invention These advantages and other advantages many embodiments and application in be exemplified, some of which is summarized by the following and runs through This specification.

Brief description

Figure 1A -1B shows polynucleotides with fluorescent marker and when analyzing such polynucleotides using nano-pore Optical noise source.

Fig. 2A -2C shows this hair that quencher is used in trans- room, cis- room and both cis- room and trans- room Bright embodiment.

Fig. 3 shows the embodiment party of the invention that illumination (epi-illumination) is penetrated using protein nano hole and falling There is case metal layer to be excited on nanohole array with reducing background or TIR and FRET.

Fig. 4 shows the confocal basic module for falling and penetrating lighting system.

Fig. 5 is shown for the optical markings object in excitation nano hole array or near nanohole array and without FRET signal The element of the TIRF system of generation.

Fig. 6 is to show based on measurement includes the optical signalling of the light from multiple (multiple) optical markings objects to adjust The flow chart of the step of with nucleotide sequence.

Fig. 7 A-7C shows the embodiment using two and three fluorescent markers.

Detailed description of the invention

Although the present invention is suitable for various modifications and alternative form, these modify and mention for form details by way of example Mode in the accompanying drawings be shown and will be described in detail.It should be understood, however, that being not intended to limit the invention to Described specific embodiment.Contrary, it is intended to cover all modifications fallen within the spirit and scope of the invention, equivalent and Substitute.For example, for purposes of illustration, it is shown that specific nano-pore type of the invention and number, specific marker, FRET to, detection scheme, manufacturing method.It should be understood, however, that present disclosure is not intended to be restricted in this respect , it is because can use other kinds of nano-pore, the array of nano-pore and other manufacturing technologies implement to be discussed herein The various aspects of system.Many available references well known within the skill of those ordinarily skilled are seen for the guidance of various aspects of the present invention Document and paper, including, for example, Cao, Nanostructures&Nanomaterials (Imperial College Press, 2004)；Levinson, Principles of Lithography, the second edition (SPIE Press, 2005)；Doering and Nishi is edited, Handbook of Semiconductor Manufacturing Technology, the second edition (CRC Press,2007)；Sawyer etc., Electrochemistry for Chemists, the second edition (Wiley Interscience, 1995)；Bard and Faulkner, Electrochemical Methods:Fundamentals and Applications, the Two editions (Wiley, 2000)；Lakowicz, Principles of Fluorescence Spectroscopy, the third edition (Springer,2006)；Hermanson, Bioconjugate Techniques, the second edition (Academic Press, 2008) Deng relevant portion is incorporated herein by reference.

The present invention relates to for using the method and dress of nano-pore and optical detection analysis of polynucleotide DNA, RNA etc. It sets.More particularly, it relates to determine the nucleotide of one or more of polynucleotides using nano-pore and optical detection Sequence.In one aspect, the present invention relates to and including inhibit and/or eliminate to come from it is expected by using non-fluorescent quencher Detection zone outside the fluorescence signal of fluorescent marker that is excited, the optical noise in signal to reduce collection.Figure 1A and 1B It schematically shows and is solved the problems, such as by this respect of the invention.In some embodiments based on optical nano-pore sequencing In, the polynucleotides being such as labeled as shown in (100) are generated by the target polynucleotide that can be obtained from sample.As institute It presents, a part of the nucleotide of the polynucleotides (100) of label has been attached (for example) two fluorescent markers " f " (102) or one in " g " (104).

Labeled polynucleotides (100) and/or the polynucleotides similarly marked by transposition by nano-pore, such as by The nano-pore that nano-pore (106) in immobilon-p (108) indicates.It is described more fully below, although nano-pore can have It by various forms and forms, but be used to realize essentially identical function, that is, by the nucleotide of marker polynucleotides It is limited to successively move and passes through detection zone.In some embodiments, nano-pore can execute other function, including (i) make Single stranded polynucleotide can not be passed through by double-stranded polynucleotide, and/or (ii) inhibits and/or limitation fluorescent marker is in nanometer Fluorescent emission during the transport of hole.The polynucleotides (100) that Figure 1B shows label pass through nano-pore from cis- room (110) (106) transposition is to trans- room (112), and the light beam that is excited (114) irradiates, and the excitation beam (114) can be is penetrated by falling The direct irradiation of irradiation or evanescent wave irradiation.In any configuration, it is expected that detection zone, such as detection zone (124) outside a large amount of glimmering Signal object is excited so that the signal collected may include from detection zone (124) outside such as comprising that will enter nano-pore (106) region (120) of the fluorescent marker of nucleotide and the fluorescence mark of the nucleotide comprising having been moved off detection zone (124) Remember the contribution of potential many fluorescent markers in the region (126) of object.As a result, the identification of the signal (128) from collection Property sequence information faces significant challenge.In one aspect, the present invention is by providing to the single stranded polynucleotide of label with affine The non-fluorescent quencher of power is come with reducing or eliminating the fluorescence signal from the region of desired detection zone or detection zone (124) outside Solve this challenge.In other respects, the non-fluorescent quencher for having affinity to the double-stranded polynucleotide of label can be provided To reduce or eliminate the fluorescence signal in the region outside desired detection zone or detection zone.

Fig. 2A -2C shows the different embodiment party of the invention for corresponding to and wherein applying quencher in nanopore device Case: only trans- room (Fig. 2A), only cis- room (Fig. 2 B) or both cis- room and trans- room (Fig. 2 C).In fig. 2a, it shows mark The polynucleotides (200) of note reach trans- room (204) from the nano-pore (206) of cis- room (202) transposition immobilon-p (208). Being immersed in trans- room (204) is the non-fluorescent quencher (205) indicated with " Q ".Quencher of the invention is for label Single stranded polynucleotide (200) metathesis conditions under be soluble, and under the same conditions, quencher and single-stranded multicore glycosides Acid such as (200) is specifically combined without sufficient sequence.Equally, in alternative embodiments, quencher of the invention exists For being soluble under the metathesis conditions of the double-stranded polynucleotide of label, and under the same conditions, quencher with it is such Double-stranded polynucleotide combines with not having sufficient sequence-specific.It is as follows more sufficiently to explain, it is various non-fluorescence sudden Agent of going out is obtainable so that for the present invention, a variety of non-fluorescent quencher include many well-known organic dyestuff, all Such as the derivative of asymmetric cyanine dye and the conjugate of such compound and oligonucleotides and/or its analog.At this In embodiment, the selection of the type and concentration and easy bit rate of quencher defines detection zone (210).In some embodiment party In case, " detection zone " means that (it can from fluorescence signal is wherein collected with the region for forming initial data (raw data) or volume To be continuous or discontinuous), the information such as sequence information of the polynucleotides about label is determined by the initial data. In some embodiments, " detection zone " is continuous region or volume.It is glimmering in the trans- room (204) of detection zone (210) outside Signal object is substantially sudden by the quencher (205) in conjunction with the part of the polynucleotides (200) marked in trans- room (204) It goes out.In some embodiments, quencher include with based on the one or more of organic dyestuff as described more fully below The oligonucleotides or the like of quencher moieties conjugation.For example, when immobilon-p (208) is or when including opaque layer, can use The embodiment of Fig. 2A, so that the fluorescent marker in cis- room (202) is not excited substantially.In such embodiment In, for example, the volume of the detection zone of the neighbouring nanometer hole exits for leading to trans- room, is likely to be dependent on including but not limited to below Factor: the concentration of quencher in the easy bit rates of the polynucleotides of label, trans- room, quencher to the affinity of polynucleotides, Quenching efficiency of quencher etc..

Fig. 2 B shows the element essentially identical with the element in Fig. 2A, in addition to quencher (205) is arranged on cis- room (202) in.Cis- room (202) is extended in undesirable evanescent wave or similar non-radiative luminous energy and generation is excited to be collected In the case where the fluorescent marker of fluorescence signal, this configuration may be desired.Multicore in cis- room (202) with label The quencher (205) that thuja acid (200) combines reduces or eliminates such fluorescence signal.In some embodiments, selection quenching The cross section of agent (205) and nano-pore (206), so that quencher (205), which is excluded, passes through nano-pore (206) without transposition.? In some embodiments, this can be realized by using protein nano hole alpha hemolysin and quencher, and the quencher includes The conjugate with one or more of quenching compounds as described more fully below of oligonucleotides or its analog.

Fig. 2 C is shown in which the embodiment party that quencher (205) is present in cis- room (202) and trans- room (204) the two Case, this embodiment provides for describe about the embodiment of both Fig. 2A and 2B.

Fig. 3 shows the embodiment including following elements: the protein nano hole being arranged in double-layer of lipoid (302) (300)；Illumination component is penetrated in falling for fluorescent marker, is had in immobilon-p (306) and is prevented or reduced the opaque of background fluorescence Layer (308)；With the quencher (310) being arranged in trans- room (326).As above, the nucleotide with fluorescent marker (marker by " f " is indicated, such as (322)) polynucleotides (320) from cis- room (324) by nano-pore (300) transposition to trans- room (326). Oligonucleotides quencher (310) is allowing oligonucleotides quencher (328) and polynucleotides (320) dew from nano-pore (300) It is arranged in trans- room (326) under the condition (such as concentration, temperature, salinity etc.) of partial hybridization out.It can choose and receive For metre hole (300) so that the signal from fluorescent marker is suppressed during nano-pore transports, the U.S. such as Huber et al. is special Benefit is announced described in US 2016/0076091, is incorporated herein by reference.Therefore, when the nucleotide of label is in region (328) when exposing from nano-pore (300) in, they become to be not inhibited and can generate signal.For most of (With Most) the direct illumination (for example, non-FRET) of (if not all) form, when the marker of such exposing is further advanced When into trans- room (326), they will continue to emit fluorescence, to greatly contribute to the signal being collected.Have trans- Quencher in room (326) in conjunction with the polynucleotides of exposing, such transmitting can be greatly reduced, and can be defined The signal of detection zone (328), the collection from the detection zone (328) can be analyzed to give about polynucleotides (320) Nucleic acid sequence information.In some embodiments, when the polynucleotides of label are mobile passes through detection zone (328), when detecting Between section from detection zone detect the fluorescence signal from single fluorescent marker.In other embodiments, in predetermined time period It is interior, more than one fluorescence signal is collected from the more than one fluorescent marker in detection zone (328).In some embodiments, Such detection time section is less than 1 millisecond or less than 0.1 millisecond or less than 0.01 millisecond.In some embodiments, in this way Detection time section be at least 0.01 millisecond or at least 0.1 millisecond or at least 0.5 millisecond.

Quencher

Quencher of the invention include under the conditions of nano-pore sequencing (i) be substantially non-fluorescence, (ii) and single-stranded core Acid, especially single stranded DNA combine, and (iii) absorbs the excitation energy from other molecules and radiationless radiationlessly Any compound (or set of compound) to release energy.In some embodiments, quencher is also non-covalent with single stranded DNA In conjunction with.Various quenching compounds are obtainable to be used for the present invention, including but not limited to as described more fully below Common synthetic dyestuffs such as cyanine dye and xanthene dye non-fluorescence derivative.Guidance in terms of selecting quenching compound United States Patent (USP) 6,323,337 can be seen；In 6,750,024 equal bibliography, these bibliography are incorporated herein by reference.

In some embodiments, quencher can be covalently is repaired with the heavy atom (such as bromine or iodine) of known quenching fluorescence Decorations, or the single stranded DNA knot of other groups (such as nitryl group or azo group) covalent modification with known quenching fluorescence Close dyestuff.One example of the known dyestuff in conjunction with single stranded DNA is Sybr Green (Zipper et al., (2004), Nucleic Acids Research.32(12)).Nitro, bromine, iodine and/or azo group are mixed in cyanine Sybr Green structure and provided Single stranded DNA conjugated group part, the fluorescent marker that quenching is likely to be present on DNA by single stranded DNA conjugated group part Object.

In some embodiments, quencher includes bound fraction and one or more quencher moieties.Bound fraction can To include any compound of no sufficient sequence specifically in conjunction with single-chain nucleic acid.Bound fraction may include with modification The peptide or oligonucleotides of key (linkage) and/or monomer or its any one analog.Oligonucleotides and the like can lead to Duplex is crossed to be formed or combine offer and the combination of polynucleotides by non-base pairing aptamer.In some embodiments, Bound fraction includes oligonucleotides or its analog with length in the range of 6 to 60 nucleotide.Such widow's core Thuja acid or the like can be conjugated with a quencher moieties or with more than one quencher moieties.In some embodiments, with it is every The more than one quencher moieties of a oligonucleotides or the like conjugation are 2 or 3.Quencher moieties with bound fraction conjugation can phase It is same or different.In some embodiments, when bound fraction is oligonucleotides or the like, two quencher moieties just and its Conjugation, the 5 ' ends and a 3 ' ends in oligonucleotides in oligonucleotides.Few core with 2 to 3 quencher moieties Conventional connection and synthesis chemical synthesis can be used in thuja acid or the like, for example, such as disclosing in references cited herein 's.

It include to combine the quencher of single-stranded oligonucleotide that can be advantageous in cis- room, because matching in some nano-pores It can choose in configuration shown in protein nano hole (300) is sufficiently narrow so that its only transposition list in such as Fig. 3 in setting The nano-pore of chain nucleic acid；Therefore, during nano-pore transposition, nano-pore carries out removing bound quenching in practice as transposition Agent.In some embodiments, it can choose and the signal of fluorescent marker inhibited to generate (or detection) during nano-pore transports, Receiving for signal (or detectability) (such as (328) in Fig. 3) is then freely generated when leaving nano-pore and entering detection zone Metre hole.

Oligonucleotides or the like can be provided with single kind or they can be with different sequences and therefore The mixture of more than one oligonucleotides with different binding specificities or the like provides.In some embodiments, Oligonucleotides or the like is random sequence polymer (random sequence polymer)；That is, they are with given length The mixture of each possibility sequence provides.For example, such oligonucleotides or the like can be by formula for 6- aggressiveness " NNNNNN ", or " NNNNNNNN " of 8- aggressiveness is indicated, wherein N can be A, C, G or T or its analog.

Refer to that " analog " of oligonucleotides means the oligonucleotides comprising one or more of nucleotide analogs.As determined Adopted part description, " nucleotide analog " is the nucleotide of the key section that can have modification, saccharide part or base portion.It can It include but is not limited to peptide nucleic acid (PNA), lock nucleic acid (LNA) (2 '-O- methyl for exemplary oligonucleotide analog of the invention RNA), phosphorothioate oligonucleotide, bridge joint nucleic acid (BNA) etc..

In some embodiments, oligonucleotides bound fraction includes universal base；That is, they include that can replace four kinds One or more of nucleotide analogs of any one of the natural nucleotide without destroying base Thermodynamic parameters.With logical Loakes, Nucleic Acids Research, 29 (12): 2437- are described in the nucleotide analog of base characteristic In 2447 (2001), it is incorporated herein by reference.In some embodiments, oligonucleotides bound fraction includes 2 '-deoxidations Inosine, 7- denitrogenation -2 '-deoxyinosine, 2- azepine -2 '-deoxyinosine, 3- nitro-pyrrole nucleotide, 5- nitroindoline nucleotide Deng.

In some embodiments, quencher may include one and work to quench the not phase of the single stranded polynucleotide of label The combination of two or more compounds of the fluorescence signal of prestige.For example, may include can be with the multicore glycosides of label for quencher Acid forms the oligonucleotides (such as more deoxyinosines) of duplex and is individually the double-strand intercalator of quencher.Therefore, whenever When poly- deoxyinosine is in conjunction with the polynucleotides of label, the quenching intercalator is in conjunction with gained duplex and quenches from multicore glycosides The fluorescence signal of acid.

The synthetic dyestuffs of the fluorescence signal of the fluorescent marker of any polynucleotides that can detectably quench label are all It is quencher moieties acceptable for the purpose of the present invention.Specifically, as being used in the present invention, quencher moieties have performance Out with the absorption band of at least some spectra overlappings of transmitting band of the fluorescent marker on the polynucleotides of label.If there is enough Spectra overlapping, this overlapping may emit with fluorescent marker (donor) and be occurred, and fluorescent marker (donor) is than sudden The low or even high wavelength emission maxima of maximum absorption wavelength of part (receptor) of going out occurs.Energy transfer can also pass through by The transmitting of donor translates into the higher electronic state of receptor and occurs.Those of ordinary skill in the art by check the dyestuff relative to The excitation band of the emission spectrum of fluorescent marker used determines the effectiveness of given quencher moieties.

In general, the fluorescence resonance between the fluorescent marker and quencher moieties of fluorescent quenching through the invention in the present invention Energy transfer (FRET or the formation by charge-transfer complex) occurs.The spectral characteristic and electronics of donor and acceptor compound Characteristic has strong influence to the degree for the energy transfer observed, fluorescent marker and quenching portion on the polynucleotides of label / separation distance be also such.With the increase of separation distance, the degree of fluorescent quenching is reduced.

Quencher moieties can optionally be fluorescence, the maximum hair of dyestuff when condition is in conjunction with the polynucleotides with label Ejected wave length is suitably separated with the maximum emission wavelength of fluorescent marker.It is preferable, however, that when total with oligonucleotides or the like When valence is conjugated, quencher moieties are only week fluorescents, or substantially non-fluorescence.As used herein, substantially non-glimmering The expression of light, as herein in the measurement solution described in any method, the fluorescence efficiency of quencher moieties is less than or equal to 5%, preferably less or equal to 1%.In other embodiments, covalently bound quencher moieties show to be less than about 0.1, more Preferably less than about 0.01 quantum yield.In some embodiments, the fluorescence to associate with quenching oligonucleotides of the invention The fluorescence of marker relative to covalently bound quencher moieties in the absence of with identical fluorescent marker association it is identical Oligonucleotides is quenched more than 50%.In another embodiment, fluorescent marker is relative to unlabelled oligonucleotides quilt Quenching is more than 90%.In another embodiment again, nucleic acid staining is quenched relative to unlabelled oligonucleotides to be more than 95%.

In some embodiments, quencher moieties can be pyrene, anthracene, naphthalene, acridine, talan, indoles or benzindole, Oxazole or benzoxazoles, thiazole or benzothiazole, 4- amino -7- nitro benzo -2- oxa--l, 3- diazole (NBD), cyanine, carbonyl Cyanine, quinolone (carbostyryl), porphyrin, salicylate (salicylate), Anthranilate (anthranilate), Azulene (azulene), perylene (perylene), pyridine, quinoline, cumarin (including Hydroxycoumarin With aminocoumarin (aminocoumarin) and its fluorination and sulfonated derivative) (U.S. Patent No. 5 of such as Gee et al., Described in U.S. Patent No. 5,696,157 (1997) of No. 830,912 (1998) and Wang et al., it is incorporated by reference into), Poly- azepine indacene (polyazaindacene) (such as the U.S. Patent No. of Haugland et al. 4,774,339 (1988)； U.S. Patent No. 5,187,288 (1993) of Kang et al.；The U.S. Patent No. of Haugland et al. 5,248,782 (1993)；U.S. Patent No. 5,274,113 (1993) of Kang et al.；The U.S. Patent No. of Kang et al. 5,433,896 (1995)；The U.S. Patent No. 6,005,113 (1999) of Wu et al., is all incorporated by reference into), xanthene, oxazines or benzo Oxazines, carbazine (U.S. Patent No. 4,810,636 (1989) of Corey, be incorporated by reference into) or Fluorenone (phenalenone) or benzfluorenone (the benzphenalenone) (U.S. Patent No. of Babb et al. 4,812,409 (1989), it is incorporated by reference into).

In other embodiments, the quencher moieties of substantially non fluorescent dye particularly include azo dyes (such as DABCYL or DABSYL dyestuff and their analogue), triarylmethane dye such as malachite green or phenol red, 4 ', 5z- The fluorescein (U.S. Patent No. 4,318,846 (1982)) that diether replaces, or (PCT is international for asymmetric cyanine dye quencher Apply WO 9937,717 (1999)).

In the embodiment that quencher moieties are xanthenes, synthetic dyestuffs are optionally fluorescein, paramethylaminophenol (rhodol) (U.S. Patent No. 5,227,487 (1993) of Haugland et al., be incorporated by reference into) or rhodamine.Such as this What text used, fluorescein includes benzo-or dibenzo fluorescein (dibenzofluorescein), half naphthofluorescein (seminaphthofluorescein) or naphthofluorescein (naphthofluorescein).Similarly, as used herein, Paramethylaminophenol include half naphthols Luo Dan glimmering (seminaphthorhodafluor) (U.S. Patent No. 4 of Haugland et al., It 945, No. 171 (1990), is incorporated by reference into).Xanthene includes fluorinated derivatives (the international publication WO 97/ of xanthene dye No. 39064, Molecular Probes, Inc. (1997) are incorporated by reference into) and xanthene dye sulfonated derivative (state Border announces WO 99/15517, and Molecular Probes, Inc. (1999) are incorporated by reference into).As used herein, Oxazines includes different pheno oxazolone (resorufm), amino oxazinone (aminooxazinone), diamino oxazines (diaminooxazine) and its benzo replace analog.

In a further embodiment, quencher moieties are the substantially non-fluorescence derivatives of 3- and/or 6- amino xanthene, It is replaced in one or more amino nitrogen atoms by aromatics or heteroaromatic ring system, for example, such as United States Patent (USP) 6,399,392 Described in, which is incorporated herein by reference.These quenchers usually have the absorption maximum value higher than 530nm, tool There is little or no observable fluorescence, and effectively quench the photism transmitting of wide spectrum, such as by chemiluminescent agent, phosphorescence The luminescence emissions of body or Fluorophore emission.In one embodiment, quencher is the rhodamine replaced.In another implementation In scheme, quenching compound is the paramethylaminophenol replaced.

Still in other embodiments, quencher moieties, which may include, is known as Black Hole described in following patent Quenchers^TMOne or more of non-fluorescent quencher of compound (BHQ): 7,019,129；7,109,312；7,582, 432；8,410,025；8,440,399；8,633,307；8,946,404；9,018,369；Or 9,139,610；These patents are logical It crosses and is incorporated herein by reference.

The other quencher moieties of following discloses, these quencher moieties pass through reference United States Patent (USP) 6,699,975；6,790, 945 and 8,114,979 are incorporated herein.

Optical signalling detection

In some embodiments, wherein excitation beam delivering and optical signalling are collected and are penetrated by falling of occurring of single object lens Lighting system can be used directly to the donor on the marker or nano-pore on irradiation polymer analysis object.For of the invention It is confocal fall penetrate lighting system basic module it is shown in Figure 4.Excitation beam (402) is directed to dichroscope (dichroic) (404) it and reaching on object lens (406), object lens (406) focus excitation beam (402) on (410) to laminar films (400), Middle marker is directly excited to emit optical signalling such as fluorescence signal, or is excited indirectly by FRET interaction To emit optical signalling.Such optical signalling is collected by object lens (406) and is oriented to dichroscope (404), dichroic Mirror (404) is selected such that it passes through the light of optical signalling (411) but the light of reflected excitation light beam (402).Optical signalling (411) By lens (414), lens (414), which are focused, to be passed through pin hole (416) and focuses on detector (418).When optics is believed When number (411) include from the fluorescence signal of more than one fluorescent marker, can provide other optical module, optical filter, Beam splitter, monochromator etc., for further separating from the different fluorescence signals from different fluorescent markers.

In some embodiments, the marker on nucleotide, which can be used, similar with equipment shown in Fig. 5 such as exists Review of the Scientific Instruments, 81:014301 (2010) of Soni et al.；And in U.S. Patent Publication Equipment described in 2012/0135410 (being incorporated herein by reference) is excited by evanscent field.In the device, in nano-pore Or the very narrow second Room on the trans- side of nanohole array allows evanscent field to extend from the surface of following glass slide, To establish detection zone at the entrance and exit of nano-pore, so that including from more with the associated each optical measurement of nano-pore In the contribution for the nucleotide that one marks.Hole array (500) (it may include the protein nano hole being inserted in double-layer of lipoid) can To be formed in silicon nitride layer (502), the silicon nitride layer (502) can have thickness in the range of 20-100nm.Nitrogen SiClx layer (502) can be formed on silicon supporting layer (503).Second Room (506) can by silicon nitride layer (502), determine second The silicon dioxide layer (504) of the height of room (506) and the surface (508) of glass slide (510) are formed.Silicon dioxide layer (504) it can have the thickness within the scope of 50-100nm.The desired of silicon nitride layer (502) is extended across from surface (508) Evanscent field (507) can be made by direct beam (512) Lai Jianli at the appropriate angle relative to glass slide (510) TIR must occur.In order to drive the polynucleotides analyte of label by array (500), can be established in the first room (516) suitable Formula (-) condition, and trans- (+) condition can be established in second Room (506), wherein electrode is operably coupled to the first He Second Room (506 and 521).

It is determined by the sequence of mixed optical signalling

In some embodiments, it can measure from a series of optical signallings for differentiating confined area, wherein each light Learning measurement, (nucleotide exists comprising the more than one component signal (component signal) from different neighbouring nucleotide Sequence in polynucleotides cannot determine by single measurement, because, such as component signal generates in diffraction limited region). In these cases, to the time series (time for generating optical measurement based on optical nano-pore analysis (i) of polynucleotides Series), which includes the overlapping contribution of the sequence of the nucleotide from more than one label, so that being difficult to (if impossible) determines the sequence of the nucleotide from single measurement；And (ii) for nucleotide by selecting to produce The optical markings object of raw differentiable signal, optical measurement can be split into the different marks on different types of nucleotide In the contribution for remembering object, this allows to be overlapped measurement and is converted into sequence information.

In one aspect, method of the invention can be implemented by following steps: polynucleotides transposition (a) being passed through nanometer Hole, wherein different types of nucleotide of polynucleotides generates the different optical markings substance markers of differentiable optical signalling, And wherein nucleotide is limited to successively move through detection zone by nano-pore, and the detection zone includes more than one nucleotide, Wherein detection zone is characterized in that there is no the quenchers in conjunction with polynucleotides；(b) when polynucleotides pass through detection zone, inspection Survey the time series of the optical signalling from nucleotide；(c) optical signalling from different types of nucleotide is distinguished；And (d) sequence of nucleotide is determined according to the time series of the optical signalling of the differentiation from polynucleotides.

According to the present invention, when the polynucleotides transposition of label passes through nano-pore and its relevant detection zone, optical measurement Time sequencing collection (time-ordered set) be recorded.It include the marker from neighbouring nucleotide from each optical measurement Contribution in the sense that say, be overlapping in the optical measurement at neighbouring time point.Thus, for example, if three nucleotide are every A time point generates signal (for example, B, C and D... of polynucleotides-A- (B-C-D)-... are moved from left to right and passed through detection Area), and if a nucleotide leaves detection zone between continuous measurement and another nucleotide enters detection zone (by including Number indicate) (for example, A enter and D leave :-(A-B-C)-D...), then continuous optical measurement twice will comprising come from phase same core The contribution of thuja acid (in this example, measuring twice all includes the contribution from B and C).Above example is easy based on polynucleotides The very simplified model that position passes through nano-pore；However, the concept of continuous overlapping optical measurement is applicable to more complicated multicore The description of thuja acid transposition.

Since the transmitting of the nucleotide of the more than one not isolabeling at nano-pore is originated from same resolution confined area, Relative position information (especially sequence information) about nucleotide cannot be determined by single optical measurement.However, due to weight The use of marker that is folded and generating polynucleotide signal, in some embodiments, when sequence information is split into core When the more than one time sequencing of thuja acid specific signals is concentrated, sequence can be determined by the time sequencing collection that optical signalling measures Information.Similar to middle using to reconstruct those of target polynucleotide sequence algorithm according to hybridization data in sequencing by hybridization (SBH) Algorithm can be used to reconstruct here target polynucleotide, such as United States Patent (USP) 5,002,867；The Biophys of Timp et al., J., 102:L37-L39 (2012) etc., is incorporated by reference into.Under the condition of optical detection feelings: (i) time sequencing overlapped signal The limitation that they are separated into polynucleotide component with signal and (ii) significantly simplifies determining step.

Fig. 6 shows the naive model based on nano-pore transposition and determines nucleotide by the time sequencing collection of overlapping optical signal The embodiment of the step of sequence information.The naive model in each time step in addition to polynucleotides enter it is assumed that (receive Metre hole and when being left from nano-pore) optical measurement respectively contain the signal contribution from equal number of nucleotide (in Fig. 6 Referred to as " n tuple " indicates that measurement will include the contribution from n nucleotide).It should be appreciated that due to the part of easy bit rate Variation, the linearly moving deviation of nucleotide and other similar phenomenons, more complicated model can permit the tribute in each measurement The number for offering nucleotide is different.That is, in some embodiments, can have in the optical measurement of different time from different numbers The contribution of purpose nucleotide.In some embodiments, different number of nucleotide sorts along the section of target polynucleotide.Figure It determines that step assumes that the polynucleotides of label have passed through nano-pore shown in 6, and has been obtained for the time of optical measurement Ordered set, including optical signalling is separated into polynucleotide signal (600).Polynucleotides enter and leave by differently Processing, because certainly existing different number of nucleotide in the detection when entering and leaving.In this embodiment, it is assumed that Initial and last optical measurement under these conditions allows initially and last nucleotide is directly according to its nucleotide Property signal determine.In other embodiments, the preparation for the nucleotide of the label of analysis may include will be more than one pre- The end or two ends for calibrating the such polynucleotides marked of nucleotides inserted of note, for generating optical signalling Known array determines the purpose of step with auxiliary sequencel.The nucleotide of such predetermined labels will be similar to that Ion Torrent or 454 sequencings, such as the critical sequences in United States Patent (USP) 7,575,865, the patent are incorporated by reference into.

Fig. 6 is returned to, when determining that step starts, time index i is arranged to zero；In the candidate sequence of current time i Index j is arranged to 1 (602)；And check the initial n tuple (604) of polynucleotide time sequencing optical signalling collection.This The inspection of sample includes determining the n tuple with the consistent all possible nucleotide of measurement according to the measurement in time i first, so Determine which is correctly be overlapped with candidate sequence Si according to these n tuples afterwards.New candidate sequence Si+1 by with measure consistent collection The respective n tuple that is correctly overlapped form (and sequence Si is extended) (606).The candidate sequence Si+1 newly extended is stored, And it gives and is updated (608) in the index Ji+1 of the number of the candidate sequence of time i+1.The step is repeated, until having checked Each candidate sequence Si (610), and similar inspection is carried out in each time i, it is concentrated until having had checked time sequencing Each optical measurement.

Nano-pore and nanohole array

It can be solid nano hole, protein nano hole for nano-pore of the invention or comprising being configured in solid film or class As protein nano hole or organic nanotube such as carbon nanotube or graphene nano pipe in frame hybridized nanometer hole.Nanometer The important feature in hole includes limitation polynucleotides analyte, the polynucleotides such as marked, so that their monomer leads in order It crosses signal and generates area (or equally, lasing region or detection zone etc.).That is, nano-pore limits polynucleotides analyte such as multicore glycosides The movement of acid, so that nucleotide in turn passes through detection zone (or lasing region).In some embodiments, the other function of nano-pore It can include that (i) pass through single-chain nucleic acid can not by double-strandednucleic acid or equivalent macromolecular, and/or (ii) is limited on nucleotide Fluorescent marker so that fluorescence signal generate be suppressed or guide so that it is not collected.

In some embodiments, the nano-pore used in conjunction with methods and apparatus of the present invention provides in an array manner, Such as array of nano-pore cluster can be regularly arranged on flat surfaces.In some embodiments, cluster is each in list In only resolution confined area, the optical signalling of the nano-pore from different clusters is enabled to be used Systems for optical inspection It distinguishes, but the optical signalling of the nano-pore in same cluster different is surely assigned to this by used Systems for optical inspection Specific nano-pore in cluster.

Solid nano hole can be manufactured with multiple material, including but not limited to silicon nitride (Si₃N₄), silica (SiO₂) Deng.(these are disclosed in following exemplary bibliography for analyzing the manufacture using the nano-pore of such as DNA sequencing and operating Bibliography is incorporated by reference into): Ling, United States Patent (USP) 7,678,562；The United States Patent (USP) 7,397,232 of Hu et al.； The United States Patent (USP) 6,464,842 of Golovchenko et al.；The United States Patent (USP) 5,798,042 of Chu et al.；The U.S. of Sauer et al. Patent 7,001,792；The United States Patent (USP) 7,744,816 of Su et al.；The United States Patent (USP) 5,795,782 of Church et al.；Bayley Et al. United States Patent (USP) 6,426,231；The United States Patent (USP) 7,189,503 of Akeson et al.；The United States Patent (USP) 6 of Bayley et al., 916,665；Akeson et al., United States Patent (USP) 6,267,872；The U.S. Patent Publication 2009/0029477 of Meller et al.； The international patent publications WO2009/007743 of Howorka et al.；The international patent publications WO2011/067559 of Brown et al.； The international patent publications WO2009/020682 of Meller et al.；The international patent publications WO2008/ of Polonsky et al. 092760；The international patent publications WO2010/007537 of Van der Zaag et al.；Yan et al., Nano Letters, 5 (6): 1129-1134(2005)；Iqbal et al., Nature Nanotechnology, 2:243-248 (2007)；Wanunu et al., Nano Letters,7(6):1580-1585(2007)；Dekker,Nature Nanotechnology,2:209-215 (2007)；Storm et al., Nature Materials, 2:537-540 (2003)；Wu et al., Electrophoresis, 29 (13):2754-2759(2008)；Nakane et al., Electrophoresis, 23:2592-2601 (2002)；Zhe et al., J.Micromech.Microeng.,17:304-313(2007)；Henriquez et al., The Analyst, 129:478-482 (2004)；Jagtiani et al., J.Micromech.Microeng., 16:1530-1539 (2006)；Nakane et al., J.Phys.Condens.Matter,15R1365-R1393(2003)；DeBlois et al., Rev.Sci.Instruments, 41 (7):909-916(1970)；Clarke et al., Nature Nanotechnology, 4 (4): 265-270 (2009)；Bayley Et al., U.S. Patent Publication 2003/0215881 etc..

In some embodiments, the present invention includes having one or more shading layers, that is, one or more opaque The nanohole array of layer.In general, the sheeting of nanohole array silicon, silicon nitride, silica, aluminium oxide etc. manufactures, They are easy to propagate light, especially in used thickness, such as are easy to propagate light less than 50-100nm.For the electricity of analyte Detection is learned, this is not a problem.However, passing through battle array based in optical detection by the molecule of the label of nano-pore in transposition The light that biographies are broadcast always excites the material outside expected reaction site, therefore generates optical noise, such as carries on the back from non-specificity The optical noise of scape fluorescence, from the fluorescence of marker of molecule etc. for not yet entering nano-pore.In one aspect, of the invention There is the nanohole array for one or more shading layers for reflecting and/or absorbing the light from excitation beam by providing, from And the ambient noise for reducing the optical signalling generated at anticipation reaction relevant to the nano-pore of array site is asked to solve this Topic.In some embodiments, this allows the optical markings object in anticipation reaction site to be excited by direct irradiation.One In a little embodiments, opaque layer can be metal layer.Such metal layer may include Sn, Al, V, Ti, Ni, Mo, Ta, W, Au, Ag or Cu.In some embodiments, such metal layer may include Al, Au, Ag or Cu.Still in other embodiments In, such metal layer may include aluminium or gold, or can only include aluminium.The thickness of opaque layer can be extensively varied, And depend on constituting the physics and chemical characteristic of the material of this layer.In some embodiments, the thickness of opaque layer can be At least 5nm or at least 10nm or at least 40nm.In other embodiments, the thickness of opaque layer can be from 5-100nm In the range of；In other embodiments, the thickness of opaque layer can be in the range of from 10-80nm.Opaque layer is not required to Stop and (reflect or absorb) 100% light from excitation beam.In some embodiments, opaque layer can stop At least 10% incident light from excitation beam；In other embodiments, opaque layer can stop at least 50% The incident light of self-excitation light beam.

Opaque layer or coating can be fabricated on solid film by various techniques known in the art.Packet can be used The material for including chemical vapor deposition, electro-deposition, extension, thermal oxide, physical vapour deposition (PVD) (including evaporating and sputtering), casting etc. is heavy Product technology.In some embodiments, atomic layer deposition, such as United States Patent (USP) 6,464,842 can be used；Wei et al., Small, 6 (13): 1406-1414 (2010) is incorporated by reference into.

In some embodiments, the channel or hole of 1-100nm can be by solid state substrate, and generally planar matrix is such as Film and formed, analyte such as single stranded DNA is induced transposition by the channel or hole.In other embodiments, 2-50nm Channel or hole formed by matrix；And still in other embodiments, 2-30nm or 2-20nm or 3-30nm or 3- The channel or hole of 20nm or 3-10nm is formed by matrix.The solid-state approach for generating nano-pore provides robustness and durability And adjustment nano-pore size and shape ability, on wafer-scale manufacture high density nanohole array ability and base Excellent machinery, chemically and thermally characteristic, and the possibility with the integration of electrically or optically sensing technique are compared in the system of lipid Property.On the other hand, by routine protein engineering method, biological nano hole provides reproducible narrow drilling or chamber, especially exists Drilling or chamber within the scope of 1-10nm, and the physically and/or chemically characteristic for customizing nano-pore technology and for direct Ground or the technology for being attached group or element such as fluorescent marker (it can be FRET donor or receptor) indirectly.Protein nano Hole often relies on exquisite double-layer of lipoid and obtains mechanical support, and the manufacture in the solid nano hole with accurate dimension is still It is challenging.In some embodiments, it can combine in solid nano hole to be formed and these is overcome to lack with biological nano hole Some so-called " hydridization " nano-pores in point, to provide the accuracy of biological porin and the stabilization in solid nano hole Property.For optical reading technique, hybridized nanometer hole provides the exact position of nano-pore, and which greatly simplifies data acquisitions.

In some embodiments, cluster can also be by being arranged in protein nano hole by the immobilon-p branch comprising hole array It is formed in the double-layer of lipoid of support.For example, such array may include manufacture (such as brill, etching etc.) in solid phase support object Hole.The geometry of this some holes can change according to used manufacturing technology.In some embodiments, each in this way Hole be associated with or be surrounded by it with individual confined area of differentiating；However, in other embodiments, more than one hole can be located In in same resolution confined area.Although these regions are typically due to conventional manufacturing method and substantially the same, different clusters it Between the cross-sectional area in hole can be extensively varied and can be identical or can be different.In some embodiments, hole has In the smallest linear dimension (such as being diameter in the case where circular hole) from 10nm to 200nm in range, or have from about 100nm²To 3 × 10⁴nm²Area in range.Double-layer of lipoid can be arranged across hole.For example, by being controlled during inserting step The concentration in protein nano hole processed, thus it is possible to vary the distribution in the protein nano hole in each hole.In such embodiments, nano-pore Cluster may include random number nano-pore.It include one in some embodiments in the radom insertion hole of protein nano hole The cluster in a or more hole has the protein nano number of perforations for being averagely greater than zero；In other embodiments, such cluster has Protein nano number of perforations greater than 0.25；In other embodiments, such cluster has the protein nano hole count greater than 0.5 Mesh；In other embodiments, such cluster has the protein nano number of perforations greater than 0.75；In other embodiments, this The cluster of sample has the protein nano number of perforations greater than 1.0.

In some embodiments, methods and apparatus of the present invention includes immobilon-p, such as SiN film, with hole array It extends there through, the hole array provides the company between the first Room and second Room (also sometimes referred to as " cis- room " and " trans- room ") Lead to and supports double-layer of lipoid on the surface towards second Room or trans- room.In some embodiments, in such immobilon-p The diameter in hole can be in the range of 10nm to 200nm, or in the range of 20nm to 100nm.In some embodiments, Such immobilon-p further includes the protein nano hole being inserted into double-layer of lipoid, and the protein nano hole is inserted in the bilayer In the region of the surface upper spanning hole towards trans- room.In some embodiments, these protein nano holes use this paper institute The technology of description is inserted into from the cis- side of immobilon-p.In some embodiments, such protein nano hole has and α hemolysin The same or similar structure because it includes bucket or drilling along axis, and an end have " cap " structure and There is " stem " structure in another end (using from Song etc., the term of Science, 274:1859-1866 (1996)). In some embodiments using such protein nano hole, the insertion to double-layer of lipoid causes protein nano hole to be oriented to So that its cap structure is exposed to cis- room and its stem structure is exposed to trans- room.

In some embodiments, the present invention can use the hybridized nanometer hole of cluster, be particularly used for polynucleotides Based on optical nano-pore sequencing.Such nano-pore includes solid-state aperture (orifice) or hole, and protein biology sensor is such as Protein nano hole is stably inserted into solid-state aperture or hole.Electrically charged polynucleotides can pass through routine protein engineering technology It is attached to protein nano hole (such as alpha hemolysin), the electric field that application hereafter can be used guides in protein nano hole to solid-state In hole in film.In some embodiments, the hole in solid state substrate is selected as being slightly less than albumen, to prevent its transposition logical Via hole.On the contrary, albumen will be embedded in solid-state aperture.

Solid nano hole or synthesis nano-pore can be produced in many ways, exemplified by bibliography as referenced above 's.In some embodiments, helium ion microscope can be used and drill out synthesis nano-pore in multiple material, such as such as Yang Deng being incorporated herein by reference disclosed in Nanotechnolgy, 22:285310 (2011).Support film material is for example One or more regions of silicon nitride (it has been machined on self-sustaining film (a free-standing membrane)) Chip is introduced in the room helium ion microscope (HIM).HIM motor controller is used for when microscope is set low to magnifying power Self-sustaining film strips are entered to the path of ion beam.Close to self-sustaining film but the region on solid matrix, adjust include focus and The light beam parameters of astigmatism (stigmation).After parameter is properly fastened, moving chip position makes self-sustaining film area Domain is concentrated on ion-beam scanning region and light beam is blanked.The visual field HIM is arranged to be enough to receive comprising entirely expected Metre hole pattern and it is enough useful ruler in following optical read-out (that is, depending on optical magnification, camera resolution etc.) It is very little (by μm in terms of).Then, once ion beam is leading to the total ion dose for being enough to remove all or most of film autofluorescence Pixel dwell time (pixel dwell time) by whole visual field, ion beam is rasterized.Then it sets the visual field to Value (less than value used above) appropriate is milled with the lithographic definition for carrying out single nano-pore or nanohole array.Pattern Pixel dwell time is arranged to cause before sample is processed determine by using calibration sample one or more predetermined The nano-pore of diameter.For region desired each of on one single chip and/or each chip for being introduced in the room HIM Repeat this complete procedure.

In some embodiments, for implementing the above method for analysis of polynucleotide (such as single stranded polynucleotide) Device generally include one group of electrode, the electrode is used to establish the electric field for crossing over laminar films and nano-pore.By by single-stranded core Acid is placed in electrolyte in the first chamber and single-chain nucleic acid is exposed to nano-pore, and first Room by placing in the chamber Negative electrode is configured as " cis- " side of laminar films.When a field is applied, negatively charged single-chain nucleic acid is captured by nano-pore, and And by the second Room on the other side of transposition to laminar films, the second Room is configured as film by placing positive electrode in the chamber " trans- " side.Transposition speed component depends on the leap of the ionic strength of electrolyte and application in the first Room and second Room and receives The voltage of metre hole.Based in optical detection, can be measured by preliminary calibration, for example, using for different voltages with not Same expection rate/nano-pore generates the preassigned product of the single-chain nucleic acid of the label of signal to select easy bit rate.Therefore, right In DNA sequencing application, easy bit rate can be selected based on the signal rate from such calibration measurement.Therefore, according to this The measurement of sample can choose the voltage across nanohole array for for example allowing or maximizing reliable Nucleotide identities.One In a little embodiments, the nucleic acid from analyzed template sample can be used (instead of preassigned sequence or except preassigned Used except sequence) carry out such calibration.In some embodiments, for example, can be sequenced operation during in real time into The such calibration of row, and the voltage that can be applied based on such measurement real time modifying, to maximize polynucleotide The acquisition of signal.

The control polynucleotides easy bit rate that pass through nano-pore is required to allow collection data, can be with from the data Obtain sequence information.Transposition speed component depends on mixed across the voltage difference (or electric field strength) of nano-pore and the reaction of the first Room The condition in object is closed, amplifying nucleic acid polynucleotides are exposed to nano-pore and (such as are disposed in and constitute the first Room in first Room In the immobilon-p of one wall).The nucleic acids polynucleotides capture rate of nano-pore depends on the concentration of such polynucleotides.In some realities It applies in scheme, the popular response mixture conditions for nano-pore sequencing can be used by the present invention, for example, 1M KCl (or wait The salt of effect, NaCl, LiCl etc.) and pH buffer system (its albumen for for example ensuring to be being used, such as protein nano hole, The invariance such as nuclease).In some embodiments, pH buffer system, which can be used, keeps pH to be substantially constant at 6.8 to 8.8 Value in range.It in some embodiments, can be in the range of from 70mV to 200mV across the voltage difference of nano-pore.? In other embodiments, the voltage difference across nano-pore can be in the range of from 80mV to 150mV.General measure can be used Technology selects operation voltage appropriate.Can be used the instrument being obtained commercially easily measure across nano-pore electric current (or Voltage).Can choose voltage difference makes easy bit rate interior in the desired range.In some embodiments, the model of easy bit rate It encloses including less than those of 1000 nucleotide/seconds speed.In other embodiments, the range of easy bit rate is from 10 cores Thuja acid/second to 800 nucleotide/seconds；In other embodiments, the range of easy bit rate is from 10 nucleotide/seconds to 600 A nucleotide/second；In other embodiments, the range of easy bit rate be from 200 nucleotide/seconds to 800 nucleotide/ Second；In other embodiments, the range of easy bit rate is from 200 nucleotide/seconds to 500 nucleotide/seconds.

Using the embodiment of mutual quenching and self-quenching marker

As mentioned above, in some embodiments, other than quencher, can also use self-quenching and mutually The fluorescent marker of quenching, to reduce the fluorescence outside those of the marker on the nucleotide for leaving nano-pore fluorescent emission Transmitting.The use of such fluorescent marker is disclosed in U.S. Patent Publication 2016/0122812, which passes through reference It is incorporated to.In some embodiments, monomer use can have following at least three kinds of states while be attached to target polynucleotide Fluorescent marker marks: the state that (i) is substantially quenched, wherein the fluorescence for the fluorescent marker being attached is by adjacent monomer Fluorescent marker quenching；For example, when the polynucleotides of label are in the conventional aqueous for studying and operating the polynucleotides In when being free, the fluorescent marker according to the present invention for being attached to polynucleotides is substantially quenched, (ii) limited space State, wherein the polynucleotides transposition marked passes through nano-pore, so that the free solution movement of the fluorescent marker of attachment (free-solution movement) or alignment be destroyed or limited so that by fluorescent marker generate it is little or no can The fluorescence signal of detection, (iii) transition state, wherein leave nano-pore (during " transition interval ") when fluorescent marker and When polynucleotides transposition passes through nano-pore, be attached to the fluorescent markers of polynucleotides from limited space status transition be quenching shape State.

Partly, which is the application to this discovery, during transition interval, (is substantially completely being marked originally In note and the polynucleotides of self-quenching) fluorescent marker can generate detectable fluorescence signal.Intention is not established this hair The limitation of existing any theory, it is believed that, the fluorescence signal generated during transition interval is due to rotatable dipole From the presence in the fluorescent marker that nano-pore exposes, this makes fluorescent marker that can briefly generate fluorescence signal, such as After direct excitation or pass through FRET.In both limited space state and quenching state, dipole is in its rotary freedom side Face is limited, to reduce or limit the number of the photon of transmitting.In some embodiments, polynucleotides are usually single-stranded The polynucleotides of polynucleotides, such as DNA or RNA, but especially single stranded DNA.In some embodiments, the present invention includes logical Overwriting generation when the fluorescent marker of attachment leaves nano-pore one at a time when polynucleotides transposition passes through nano-pore Signal come the method that determines the nucleotide sequence of polynucleotides.When leaving, each of on the polynucleotides in the solution that dissociates The fluorescent marker of attachment is quenching state from the constrained state transition in nano-pore during transition interval.In other words, one In a little embodiments, the step of method of the invention include each fluorescent marker on the polynucleotides in free solution by Constrained state transition in nano-pore excites them when being quenching state.As mentioned above, this transition interval or when Between in section fluorescent marker can emit the detectable fluorescence signal of nucleotide of the instruction attached by it.

In some embodiments, the present invention includes the application of following discovery: it can choose fluorescent marker and nano-pore, So that the fluorescent marker for being attached to monomer is forced into constrained state during polynucleotides transposition is by nano-pore, Under this state, they can not (or substantially can not) generate detectable fluorescence signal.In some embodiments, it selects Select the nano-pore with diameter in drilling or chamber from 1nm to 4nm in range；In other embodiments, selection has diameter In the nano-pore of drilling or chamber from 2nm to 3nm in range.In some embodiments, the diameter of such drilling passes through egg White nano-pore provides.In some embodiments, such nano-pore be used to that fluorescent marker be forced to enter according to the present invention Constrained state, so as to be by can not substantially generate fluorescence signal transition when fluorescent marker leaves nano-pore The fluorescence signal institute detection and identification that can be induced by it and emit.Therefore, it is attached to each of the sequence monomer of polynucleotides The fluorescent marker of monomer when its suddenly generate fluorescence signal when can close to the region of nanometer hole exits (" transition region " or " transition volume " or " detection zone ") sequentially detected.In some embodiments, more than organic fluorescent dye is used as having The fluorescent marker of the nano-pore of diameter.In some embodiments, organic fluorescent dye as at least one is selected from by Xanthones The set of ton dyestuff, rhodamine and cyanine dye composition.For determining some embodiment party of the sequence monomer of polynucleotides Case can be carried out by following steps: (a) make polymer transposition by nano-pore, wherein the monomer of polynucleotides is fluorescently labeled Substance markers, wherein nano-pore drilled in fluorescent marker be limited to that constrained state not generate substantially wherein can The fluorescence signal of detection；(b) fluorescent marker of each monomer is excited when each monomer leaves nano-pore；(c) measurement is by just The fluorescence signal generated in the fluorescent marker left in detection zone is to identify monomer attached by fluorescent marker；(d) it quenches The fluorescence signal of the fluorescent marker of excitation outside detection zone, and multicore glycosides (d) is determined according to the sequence of fluorescence signal The sequence monomer of acid.In a further embodiment, fluorescent marker is FRET pairs of receptor and the one of FRET couples or more Multiple donors are attached to the FRET in outlet on interior nano-pore.

In some embodiments, such as the fluorescence letter for meaning fluorescent marker " substantially quenching " and generating used above Number than under the same conditions but the signal that generates of when not neighbouring mutual quenching marker reduces at least 30 percent.One In a little embodiments, if the fluorescence signal ratio for meaning fluorescent marker generation " substantially quenching " used above is in identical item The signal generated under part but when not neighbouring mutual quenching marker reduces at least 50 percent.

In some embodiments, the nucleotide sequence of target polynucleotide is determined by carrying out four individual reactions, The wherein single fluorescent marker of each in four kinds of the copy of target polynucleotide different types of nucleotide (A, C, G and T) Object marks.In the version of such embodiment, the nucleotide sequence of target polynucleotide is by carrying out four kinds individually Reaction determine, wherein each use in the different types of nucleotide of the four of the copy of target polynucleotide kind (A, C, G and T) A kind of fluorescent marker marks, and other nucleotide on the identical target polynucleotide are marked with the second fluorescent marker simultaneously Note.For example, if the first fluorescent marker is attached to the A of target polynucleotide in the first reaction, in first reaction In the second fluorescent marker be attached to C, G and T (that is, being attached to " nucleotide of non-A ") of target polynucleotide.Similarly, Continue the example, in the second reaction, the first marker is attached to the C of target polynucleotide, and the second fluorescent marker is attached It is connected to A, G and T (being attached to " non-C " nucleotide) of target polynucleotide.For nucleotide G and T, and so on.

For the subset of nucleotide type, identical tagging scheme can be expressed according to general term；Therefore, above real In example, in the first reaction, the first fluorescent marker is attached to A, and the second fluorescent marker is attached to B；It is anti-second Ying Zhong, the first fluorescent marker is attached to C, and the second fluorescent marker is attached to D；In third reaction, the first fluorescence Marker is attached to G, and the second fluorescent marker is attached to H；And in the 4th reaction, the first fluorescent marker is attached It is connected to T, and the second fluorescent marker is attached to V.

In some embodiments, polymer such as polynucleotides or peptide can be labeled, wherein single fluorescent marker is attached It is connected to the monomer of single kind, for example, each T (or substantially each T) fluorescent marker of polynucleotides, such as cyanine dye Material label.In such embodiments, the set of the fluorescence signal from polynucleotides or sequence can be formed for specific The feature or fingerprint of polynucleotides.In some such embodiments, such fingerprint can provide or can not provide foot To determine the information of sequence monomer.

In some embodiments, the invention is characterized in that being used as mutually quenching the fluorescent dye or mark of the member of set Object is remembered to mark the essentially all monomer of polynucleotides analyte.Term about label polynucleotides analyte is " substantially Use all ", which refers to, recognizes that chemical labeling techniques and enzyme labelling technique are usually less than 100% effectively.In some embodiments In, " substantially all " means that at least the 80% of whole monomers have the fluorescent marker of attachment.In other embodiments, " substantially all " means at least 90% fluorescent marker with attachment in whole monomers.In other embodiments, " basic Upper whole " means at least 95% fluorescent marker with attachment in whole monomers.The set of the fluorescent dye mutually quenched has Have following characteristic: (i) each (each) member quenches the fluorescence of each (every) member (for example, by FRET or by quiet State or contact mechanism), and (ii) each member generates different fluorescence letters when excited and when being in non-quenching state Number.That is, if mutually quenching set be made of two kinds of dyestuffs D1 and D2, (i) D1 be self-quenching (such as by with another The quenching of D1 molecule contacts) and it quenches (such as pass through contact quench) by D2 and (ii) D2 is that self-quenching (such as passes through Quenched with another D2 molecule contacts) and its (such as by contact quench) is quenched by D1.About selection for mutually quenching The guidance of the fluorescent dye or marker of set is found in below with reference to document, these bibliography are merged into this by reference Text: Johansson, Methods in Molecular Biology, 335:17-29 (2006)；Marras et al., Nucleic Acids Research, 30:el22 (2002) etc..In some embodiments, the member of mutually quenching set includes organic Fluorescent dye, component or competent stack of part such as aromatic ring structure interact.Fluorescent dye or marker it is exemplary Mutually quenching set can be selected from rhodamine, fluorescein(e) dye and cyanine dye.In one embodiment, mutually quenching Set may include rhodamine TAMRA and fluorescein(e) dye FAM.In another embodiment, can by from by with The mutual quenching for selecting two or more dyestuffs to form fluorescent dye in the group of lower composition is gathered: Oregon Green 488, Red-the X of fluorescein-EX, fluorescein isothiocynate, rhodamine (Rhodamine Red-X), Sulforhodamine B, calcein, Fluorescein, rhodamine, one or more BODIPY dyestuff, texas Red (Texas Red), the green (Oregon in Oregon Green) 514 and one or more Alexa Fluor.Representative BODIPY dyestuff includes BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY581/591, BODIPY TR, BODIPY 630/650 and BODIPY 650/665.It is representative Alexa Fluor includes Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488、Alexa Fluor 500、Alexa Fluor 514、Alexa Fluor 532、Alexa Fluor 546、 Alexa Fluor 555、Alexa Fluor 568、Alexa Fluor 594、Alexa Fluor 610、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、Alexa Fluor680、Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790.

As described above, in some embodiments, the sequence monomer of target polynucleotide is by individually being reacted (every kind A kind of reaction of monomer) it determines, wherein each of copy of target polynucleotide different types of monomer is mutually quenched or from sudden The fluorescent label to go out.In other embodiments, the sequence monomer of target polynucleotide is by individually being reacted (every A kind of kind of reaction of monomer) determine, wherein the different types of monomer of each of copy of target polynucleotide by selected from it is same mutually Quench the different fluorescent labels mutually quenched of set.Wherein mutually quenching reality of the set comprising only two kinds of dyestuffs It applies in scheme, then the dye marker that the monomer (such as monomer X) selected is mutually quenched with first, and each other kinds of list The dye marker that body (that is, non-monomer X) is mutually quenched with second from identity set.Therefore, the step of embodiment produces The sequence for the fluorescence signal that two are not planted, an instruction monomer X are given birth to, and another indicates non-monomer X.

In some embodiments, single fluorescent marker can be used (for example, being attached to comprising more than one type list On the monomer of the single kind of the polynucleotides of body), when it is attached to (same type of) on polynucleotides neighbouring monomer, It is self-quenching when the neighbouring nucleotide of such as polynucleotides.Exemplary self-quenching fluorescent marker includes but is not limited to that Russia strangles Ridge is green 488, fluorescein-EX, FITC, the red-X of rhodamine, Sulforhodamine B, calcein, fluorescein, rhodamine, BODIPY And texas Red, such as they are disclosed in Molecular Probes Handbook, and 2010 in the 11st edition.

Using the embodiment of two or three of optical markings object

In some embodiments, the different optical markings substance markers of as little as two different kinds of nucleotide, it is described Different optical markings objects is generated for the nucleotide of the type selected in both the sense strand of target polynucleotide and antisense strand Differentiable optical signalling.For example, the analog that the C and T on the complementary strand of each target polynucleotide can be labeled replaces, The marker of middle C and T analog can generate different optical signallings.Then by make label chain transposition by nano-pore come Optical signature is generated, wherein the nucleotide of the chain is restricted to pass sequentially through optical detection zone, in the optical detection zone In, their marker is caused generation optical signalling.It in some embodiments, will be from both sense strand and antisense strand The information of optical signature combines the nucleotide sequence to determine target polynucleotide.

In some embodiments, the nucleotide by selection type of target nucleotide is anti-using the extension of nucleic acid polymerase The nucleotide analog being labeled in answering replaces.The chain transposition of the label of target polynucleotide is set to pass through nano-pore, the nano-pore The nucleotide of chain is limited so as to successively mobile by optical detection zone, in optical detection zone the nucleotide of chain be excited so that it Generate optical signalling.The set of the optical signalling of individual chain is referred to herein as the optical signature of the chain.In some implementations In scheme, in chain and its complementary strand (i.e. sense strand and antisense strand) for example by the connected situation of hair clip adapter, individually Optical signature may include the optical signalling of the optical markings object on the nucleotide from both sense strand and antisense strand.At other In embodiment, the different chains of target polynucleotide can individually generate two different optical signatures, described two different Optical signature can be combined or together for analyzing, as mentioned above.The chain of such independent analysis can produce After raw optical signature for example by using molecular label (its can be for example with known position, length and sequence pattern with And allow to be easy the oligonucleotide segments that associated diversity is attached to target polynucleotide) associated.As noted below, this The optical signature of invention may include mixed optical signalling, because the signal detected in each detection interval may include From in the contribution for differentiating the more than one optical markings object emitted in confined area or volume；That is, they can (for example) be Mixed FRET signal is incorporated by reference into as described in the U.S. Patent Publication US20160076091 of Huber et al. Herein.

As mentioned above, in some embodiments, method of the invention can be implemented by following steps: (a) chain of double-stranded polynucleotide is copied, so that the nucleotide analog with different optical markings objects replaces at least two Nucleotide, to form the chain of label；(b) complementary strand of the chain is copied, so that nucleotide analog substitution is identical extremely Few two kinds of nucleotide, to form the complementary strand of label；(c) make the chain transposition of the label by nano-pore, so that the label The nucleotide of chain pass sequentially through detection zone, optical markings object is excited to generate optical signalling in the detection zone；(d) sudden The fluorescence signal of the fluorescent marker for the excitation gone out outside detection zone；(e) when the chain transposition of the label passes through nano-pore, The time series of the optical signalling from optical markings object is detected to generate the optical signature of chain；(f) make the complementation of the label Chain transposition is by nano-pore, so that the nucleotide of the complementary strand of the label passes sequentially through lasing region, the light in the lasing region Marker is learned to be excited to generate optical signalling；(g) fluorescence signal of the fluorescent marker of excitation of the quenching outside detection zone； (h) when the complementary strand transposition of the label passes through nano-pore, the time series of the optical signalling from optical markings object is detected To generate the optical signature of complementary strand；(i) according to the optical signature of the chain and the determination of the optical signature of the complementary strand The sequence of double-stranded polynucleotide.In some embodiments, two kinds of nucleotide are labeled, can be C and T, C and G, C and A, T With G, T and A or G and A.In some embodiments, pyrimidine nucleotide is labeled.In other embodiments, purine nucleotides It is labeled.In some embodiments, the nucleotide by selection type of chain is anti-in primer extend by using nucleic acid polymerase The analog dNTP of the label of the middle incorporation nucleotide by selection type is answered to mark.In other embodiments, chain It is marked by the nucleotide of selection type by mixing the analog dNTP of the nucleotide by selection type in extension Note, wherein analog dNTP is derived with orthogonal reaction functionality, is allowed different markers in subsequent reaction It is attached to different types of nucleotide.The latter labeling method is disclosed in the United States Patent (USP) 5,405,747 of Jett et al., should Patent is incorporated herein by reference.

In some embodiments, three kinds of nucleotide are labeled, may include with the first optical markings substance markers C, use Second optical markings substance markers T, and with third optical markings substance markers G and A.It in other embodiments, can following institute The group of following nucleotide is remembered in indicating: respectively with the first optical markings object and the second optical markings substance markers C and G, and using third Optical markings substance markers T and A；Respectively with the first optical markings object and the second optical markings substance markers C and A, and with third light Learn label substance markers T and G；Respectively with the first optical markings object and the second optical markings substance markers T and G, and with third optics Mark substance markers C and A；Respectively with the first optical markings object and the second optical markings substance markers A and G, and with third optics mark Remember substance markers T and C.

In some embodiments, optical markings object is produced after the energy transfer from the donor to associate with nano-pore The fluorescence acceptor molecules of raw fluorescence resonance energy transfer (FRET) signal.In some embodiments, as described further below , donor can be optical activity nano particle, quantum dot, Nano diamond etc..The specific group of acceptor molecule and donor The selection of conjunction is the design alternative of those of ordinary skill in the art.In some embodiments, (some of which is more filled below Divide ground description) in, single quantum dot is attached to nano-pore and is excited using excitation beam to issue fluorescence, the excitation The wavelength of light beam is sufficiently separated, usually lower (i.e. more blue), so that excitation beam does not have the FRET signal generated by receptor Contribution.Equally, the quantum dot for selecting its launch wavelength Chong Die with the absorption band of two acceptor molecules is to promote FRET to interact. In some embodiments, two donors can be used for each lasing region of nano-pore, wherein the launch wavelength of each donor It is selected as most preferably being overlapped from the absorption band of a different acceptor molecules.

In fig. 7, double-strand target polynucleotide (700) (SEQ ID NO:1) is by sense strand (701) and complementary antisense strand (702) it forms, " Y " adapter (704) and adapter (706) is connected into (703) to double-strand target polynucleotide using conventional method, The United States Patent (USP) 6,287,825 of the conventional method such as Weissman et al.；The U.S. Patent Publication of Schmitt et al. US2015/004468；It is incorporated herein by reference.The arm (708) and arm (710) of adapter (respectively 704 and 706) include Primer (716) and primer (718) annealing (705) are to its primer binding site.Double stranded section (712) and double stranded section (714) It may include sequence label, for example, one or two double stranded section may include the random molecular of predetermined length and composition (randomer), it can be used for being associated with again for subsequent chain, for example, to obtain the sequence of the optical signature from respective chain Information.After making primer (716) and primer (718) anneal, they can be in the dUTP class of (for example, as shown) label Like the dCTP analog of object (being shown as the marker of empty circles in the nucleotide of incorporation) and label (in the nucleotide of incorporation In be shown as the markers of solid circles) and natural unlabelled dGTP and dATP (both without unlabelled dTTP or without not The dCTP of label exists, so that analog is fully substituted in the chain of extension) in the presence of pass through nucleic acid polymerase quilt Extend (707).Being not present for marker is indicated above the nucleotide of incorporation with dash on G and A.Do not having noisy ideal In detection system, the sequences of empty circles, solid circles and dash, which will be sense strand by showing and antisense strand, leads at them Cross the good expression of the optical signature generated when the lasing region of nano-pore.

In figure 7b, the extension products (720) and extension products using the optional embodiment of three kinds of markers are shown (722).As it appears from the above, the dUTP analog of the label of incorporation is shown as empty circles, and the dCTP class of the label mixed It is shown as solid circles like object.The dATP analog and dGTP analog of the label of incorporation are shown as solid diamond.

About selecting the type of nucleotide to be marked, the type of marker and for marker to be attached to connecing for base The type of head and the guidance of the nucleic acid polymerase for extension in the presence of dNTP analog are found in following ginseng Document is examined, these bibliography are incorporated by reference into: the United States Patent (USP) 5,945,312 of Goodman et al.；The U.S. of Jett et al. Patent 5,405,747；The U.S. Patent Publication US2004/0214221 of Muehlegger et al.；Giller et al., Nucleic Acids Research, 31 (10): 2630-2635 (2003)；Tasara et al., Nucleic Acids Research, 31 (10):2636-2646(2003)；Augustin et al., J.Biotechnology, 86:289-301 (2001)；Brakmann, Current Pharmacuetical Biotechnology, 5 (1): 119-126 (2004) etc..For of the invention exemplary Nucleic acid polymerase includes but is not limited to Vent exo-, Taq, Escherichia coli (E.coli) Pol I, Tgo exo-, Klenow segment Exo-, Deep Vent exo- etc..In some embodiments, exemplary nucleic acid polymerase includes but is not limited to Vent exo- With Klenow segment exo-.Exemplary fluorescence marker for dNTP analog include but is not limited to Alexa 488, AMCA, Atto 655, Cy3, Cy5, Evoblue 30, fluorescein, Gnothis indigo plant 1, Gnothis indigo plant 2, Gnothis indigo plant 3, Dy630, Dy635, MR121, rhodamine, rhodamine green (Rhodamine Green), the green, TAMRA in Oregon etc..For dUTP analog Exemplary fluorescence marker include but is not limited to Alexa 488, AMCA, Atto 655, Cy3, Cy5, Dy630, Dy665, Evoblue 30, Evoblue 90, fluorescein, Gnothis indigo plant 1, Gnothis indigo plant 2, Gnothis indigo plant 3, MR121, Oregon be green, Green, TAMRA of rhodamine, rhodamine etc..Exemplary fluorescence marker for dCTP analog include but is not limited to Atto 655, Cy5, Evoblue 30, Gnothis indigo plant 3, rhodamine, the green, TAMRA of rhodamine etc..Exemplary fluorescence for dATP analog Marker includes but is not limited to that Atto 655, Cy5, Evoblue 30, Gnothis indigo plant 3, rhodamine are green etc..It is similar for dGTP The exemplary fluorescence marker of object includes but is not limited to that Evoblue 30, Gnothis indigo plant 3, rhodamine are green etc..It is similar for dUTP The exemplary fluorescence marker of object and dCTP analog to include but is not limited to (TAMRA, rhodamine are green), (Atto 655, Evoblue 30), (Evoblue 30, Atto 655), (Evoblue 30, Gnothis indigo plant 3), (Evoblue30, rhodamine It is green), (Gnothis indigo plant 1, rhodamine is green), (Gnothis indigo plant 2, Atto 655), (Gnothis indigo plant 3, Cy5) etc..

Fig. 7 C shows an embodiment, wherein using two kinds of markers, and is connected by hair clip adapter (730) Sense strand and antisense strand, such as taught in U.S. Patent Publication US 2015/0152492 and US 2012/0058468, It is incorporated herein by reference.By the adapter (732) of tailing and hair clip adapter (730) and target polynucleotide (700) (SEQ ID NO:1) connection.It is being denaturalized with after primer (734) annealing, extension generates extension products (735), and it includes sections (736) (it is the complementary strand of the label of chain (701)) and section (738) (it is the reverse complementary strand of the label of chain (701)).? After extension products (735) transposition passes through nano-pore and generates optical signature, target polynucleotide (700) (SEQ ID can be determined NO:1 sequence).It is optionally possible to which the sequence of hair clip (730) is selected to mix scheduled mode during extension Marker can be used for such as auxiliary section (736) end at where and section (738) starts from where by indicating Help the analysis of optical signature.

Kit

The present invention may include the kit for carrying out method of the invention.In some embodiments, kit packet Include the one or more of quenchers for being suitable for quenching the fluorescence signal generated by the fluorescent marker for being attached to target polynucleotide.Example Such as, kit can be designed to the absorption band transmitting band with fluorescent marker on target polynucleotide to the maximum extent comprising it The quencher of overlapping.In some embodiments, the quencher of kit includes the polynucleotides combination for being conjugated to quencher moieties Part.In some embodiments, polynucleotides bound fraction includes one or more of oligonucleotides or its analog.One In a little embodiments, the quencher of kit is provided with scheduled concentration.In some embodiments, selection is such scheduled dense The quencher of degree determines the volume of detection zone.

Definition

" evanscent field (evanescent field) " means non-propagating electromagnetic field；That is, it is the average value of Poynting vector The electromagnetic field for being zero.

" FRET " or "Or fluorescence resonance energy transfer " mean the donor fluorophore from excitation in ground state Acceptor fluorescence group non-radiative dipole-dipole energy transfer mechanism.Energy transfer rat in FRET interaction depends on The degree of spectra overlapping, the quantum yield of donor, donor and the acceptor transition of the absorption spectrum of the emission spectrum and receptor of donor The relative orientation and the distance between donor molecule and acceptor molecule of dipole, Lakowitz, Principles of Fluorescence Spectroscopy, the third edition (Springer, 2006).FRET interaction of special interest is that Part energy is caused to be transferred to receptor, then (its frequency is lower than the light for exciting its donor by acceptor emission as photon Frequency (i.e. " FRET signal ")) FRET interaction." FRET distance " means that FRET interaction can occur in it And FRET receptor can produce the distance between FRET donor and FRET receptor of detectable FRET signal.

" kit " refers to any delivery system for delivering the substance or reagent that carry out method of the invention.It is surveyed in reaction In the case where fixed, such delivery system includes allowing reaction reagent (such as the fluorescent marker in container appropriate being such as Fluorescent marker, fluorescent marker bridging agent, enzyme, quencher for mutually quenching etc.) and/or support substance (for example, buffer, Written explanation etc. for being measured) from a position to the system and/or change of the storage of another position, transport or delivering It closes object (diluent, surfactant, carrier etc.).For example, kit includes comprising correlated response reagent and/or support One or more attachmentes (such as box) of matter.Such content can together or individually be delivered to expected receiving Person.For example, first container may include the enzyme for measurement, and the second or more container includes the fluorescence mark mutually quenched Remember object and/or quencher.

" nano-pore " means any opening being located in matrix, allows analyte logical with scheduled or recognizable sequence Matrix is crossed, or in the case where polymer analysis object, allows the monomeric unit of polymer analysis object with scheduled or can distinguish Sequence pass through matrix.In the latter case, scheduled or recognizable sequence can be one of monomeric unit in polymer Grade sequence.The example of nano-pore includes (proteinaceous) nano-pore of albumen or the nano-pore based on albumen, synthesizes nanometer Hole or solid nano hole, and the hybridized nanometer hole in the solid nano hole including being wherein embedded with protein nano hole.Nano-pore can With the internal diameter with 1-10nm or 1-5nm or 1-3nm.The example in protein nano hole includes but is not limited to, alpha hemolysin, voltage according to Bad property mitochondrial porin (VDAC), OmpF, OmpC, MspA and LamB (maltoporin (maltoporin)), such as Rhee, M. et al., Trends in Biotechnology, 25 (4) (2007): 174-181；Bayley et al. is (referenced above )；Disclosed in the U.S. Patent Publication 2012/0055792 of Gundlach et al. is equal, it is incorporated in herein by reference.It can Using any albumen hole for allowing single nucleic acid molecules transposition.Nanometer porin can at the specific site outside hole or At the specific site for constituting the outside of one or more monomeric units of hole formation albumen (pore forming protein) It is labeled.Porin is selected from the group of such as, but not limited to albumen below: alpha hemolysin, MspA, voltage-dependent mitochondria Porin (VDAC), anthrax porin, OmpF, OmpC and LamB (maltoporin).By the way that electrically charged polymer is attached Porin is connected to realize porin to the integration in solid-state hole.After applying an electric field, electrically charged compound passes through electrophoresis It is drawn into solid-state hole.The nano-pore of synthesis or solid nano hole can be created in various forms of solid state substrates, Gu The example of state matrix includes but is not limited to silicone (such as Si₃N₄、SiO₂), metal, metal oxide (such as Al₂O₃), plastics, Glass, semiconductor material and combinations thereof.Synthesizing nano-pore can be more more stable than the bioprotein hole being located in bilayer lipid membrane.It closes It can also be created by using the carbon nanotube for being embedded in the epoxy resin that suitable matrix such as, but not limited to polymerize at nano-pore It builds.Carbon nanotube can have chemistry that is consistent and being able adequately determines and architectural characteristic.The carbon nanotube of various sizes can be obtained, Range is from one nanometer to several hundred nanometers.The surface charge of known carbon nanotube is about zero, and therefore, and nucleic acid passes through nano-pore Electrophoresis transhipment becomes simple and predictable (Ito, T. et al., Chem.Commun.12 (2003): 1482-83).Synthesize nano-pore Stromal surface can be modified by sulphation with allow albumen hole covalently be attached or make surface characteristic be suitable for optical nano survey Sequence.Such surface modification can be covalent or non-covalent.Most of covalent modifications include organic silane-deposited, most normal Scheme is described as: 1) depositing from aqueous alcohol.This is the method being easiest to for being used to prepare silylated surface.With - 5% aqueous solution of 95% ethyl alcohol is adjusted to pH 4.5-5.5 by acetic acid.Add silane under stiring to generate 2% ultimate density. After hydrolysis and silanol are formed, continues 2-5min and add matrix.Excess is rinsed out by simple dipping in ethanol Material.Silylation layer is solidificated in 110 degrees Celsius of lasting 5-10min.2) it is vapor-deposited.Silane can pass through chemical vapor deposition Method is applied in matrix under dry aprotic conditions.These methods are conducive to monolayer deposition.It is set in closed room In meter, matrix is heated to sufficiently achieve the temperature of 5mm vapour pressure.It is alternatively possible to apply vacuum, until observing that silane steams Hair.3) spin-on deposition (spin-on deposition).Spin coating application can be conducive to maximum functionalization and plane SH wave It is carried out under hydrolysising condition or under the drying condition for being conducive to monolayer deposition.In some embodiments, method of the invention is adopted With single nano-pore.In other embodiments, using more than one nano-pore.In some embodiments below, it is more than One nano-pore is used as nanohole array, and the nanohole array is commonly disposed to planar substrate such as immobilon-p In.The nano-pore of nanohole array can be spaced apart by regularly, such as with straight line pattern, or can be randomly spaced It opens.In a preferred embodiment, nano-pore is regularly spaced apart in planar solid phase matrix with straight line pattern.

" polymer " means the more than one monomer for being connected as straight chain.In general, polymer includes the list of more than one type Body for example, include A, C, G and T as polynucleotides, or as polypeptide includes more than one amino acid.Monomer may include But it is not limited to nucleosides and its derivative or the like and amino acid and its derivative and analogue.In some embodiments, Polymer is polynucleotides, and wherein nucleoside monomers are connected by phosphodiester bond or its analog.

" polynucleotides " or " oligonucleotides " are interchangeably used, and respectively mean nucleotide monomer or its analog Linear polymer.The monomer for constituting polynucleotides and oligonucleotides can be by monomer and monomer interaction such as Watson- The mode of rule of the base pairing of Crick type, base stacking, the base pairing of Hoogsteen or reversed Hoogsteen type etc. is special The opposite sex is bound to native polynucleotide.Such monomer and its internucleoside linkage can be naturally occurring, or can be its class Like object, such as naturally occurring analog or non-naturally occurring analog.Non-naturally occurring analog may include PNA, Connect key between thiophosphoric acid nucleosides, comprising the base for the linking group for allowing marker (fluorogen or haptens etc.) to be attached Deng.Whenever the processing of the requirement enzymatic of oligonucleotides or polynucleotides, is such as extended through polymerase, passes through ligase connection Whens equal, ordinarily skilled artisan will understand that, under those circumstances, oligonucleotides or polynucleotides are in any position or at certain positions Certain analogs of internucleoside linkage, saccharide part or base will not included.The size range of polynucleotides is usually from several monomers Unit such as 5-40 (polynucleotides are commonly known as " oligonucleotides " at this time) extremely thousands of monomeric units.Unless otherwise indicated Or from the context it will be evident that when polynucleotides or oligonucleotides are indicated with alphabetical (upper case or lower case) sequence, such as " ATGCCTG ", it should be appreciated that nucleotide is from left to right 5 ' → 3 ' sequence, and " A " indicates that desoxyadenossine, " C " indicate de- Oxygen cytidine, " G " indicates deoxyguanosine, and " T " indicates that thymidine, " I " indicate that deoxyinosine, " U " indicate uridine.Unless otherwise saying Bright, term and atom numbering convention will comply in Strachan and Read, 2 (Wiley- of Human Molecular Genetics Liss, New York, 1999) disclosed in those.Usual polynucleotides include be keyed by di-phosphate ester four kinds naturally Nucleosides is (for example, be used for desoxyadenossine, deoxycytidine, deoxyguanosine, deoxythymidine or their ribose pair for RNA of DNA Answer object)；However, they can also include non-natural nucleotide analog, between base, sugar or nucleosides for example including modification Key.For a person skilled in the art it is clear that the activity in enzyme has to specific oligonucleotides or polynucleotide substrate example In the case where such as requirement of single stranded DNA, RNA/DNA duplex, then for suitable group of oligonucleotides or polynucleotide substrate At selection completely in the knowledge of those of ordinary skill, especially come from paper, such as Sambrook et al., Molecular Cloning, the second edition (Cold Spring Harbor Laboratory, New York, 1989) and similar Bibliography guidance under.Equally, oligonucleotides and polynucleotides can refer to single stranded form or double-stranded form (i.e. oligonucleotides Or the duplex of polynucleotides and its respective complementary strand).Which the context that those of ordinary skill uses from term will be clear that Form is desired or whether two kinds of forms are all desired.

" primer " means natural or synthesis oligonucleotides, can make when forming duplex with polynucleotide template For nucleic acid synthesis starting point and can from its 3 ' end along template extend, to form the duplex of extension.Primer prolongs It stretches and is usually carried out with nucleic acid polymerase such as DNA or RNA polymerase.The sequence of the nucleotide added during extension is by mould The sequence of plate polynucleotides determines.Primer is usually extended through archaeal dna polymerase.Primer usually has from 14 nucleotide to 40 In a nucleotide range or the length in from 18 nucleotide to 36 nucleotide ranges.It is anti-that primer is used for various nucleic acid amplifications Ying Zhong, such as reacted using the linear amplification of single primer, or using the polymerase chain reaction of two or more primers.It is right In selection for specific application primer length and sequence guidance be it is well known to those of ordinary skill in the art, such as pass through It is proved below with reference to document: the PCR Primer:A Laboratory Manual that Dieffenbach is edited, the second edition (Cold Spring Harbor Press, New York, 2003), is incorporated by reference into.

" differentiating confined area " is that the individual on the surface of nano-pore (nanopore) or nano-pore (nanowell) array is special Sign or photo-emission source are in the region that can not be wherein distinguished by optical signal detection system.It is not intended to be bound by theory, the resolution Confined area is determined by the limit of resolution (also sometimes referred to as " diffraction limit " or " diffraction barrier ") of optical system.The limit by The wavelength and optical component of emission source determine, and can be defined by d=λ/NA, and wherein d is the minimal characteristic that can be resolved, λ is the wavelength of light, and NA is the numerical aperture for focusing the object lens of light.Therefore, whenever two or more nano-pores exist When differentiating in confined area and generating two or more optical signallings at each nano-pore, Systems for optical inspection can not area Divide or determines which optical signalling comes from which nano-pore.According to the present invention, the surface of nanohole array can be divided or carefully It is divided into and corresponds to the Non-overlapping Domain for differentiating confined area or substantially Non-overlapping Domain.Corresponding to this of resolution confined area The size of subdivision can depend on used particular optical detection system.In some embodiments, whenever photo-emission source exists When in visible spectrum, confined area is differentiated from 300nm²To 3.0 μm²In the range of；In other embodiments, it differentiates limited Region is from 1200nm²To 0.7 μm²In the range of；In other embodiments, confined area is differentiated from 3 × 10⁴nm²Extremely 0.7μm²In the range of, wherein the range of aforementioned areas is about nano-pore (nanopore) or nano-pore (nanowell) array Surface.In some embodiments, it is seen that spectrum means in the wavelength within the scope of from about 380nm to about 700nm.

It include to polynucleotides about terms such as " sequence determine " of polynucleotides, " sequencing " or " determining nucleotide sequence " Part and full sequence information determination.That is, these terms include the complete collection of four kinds of natural nucleotides A, C, G and T The sequence of the subset of conjunction, such as only A of target polynucleotide and the sequence of C.That is, these terms include in target polynucleotide One of nucleotide of four seed types, two kinds, three kinds or whole of identity, sequence and the determination of position.In some embodiment party In case, these terms include two kinds, three kinds or whole of identity, sequence in the nucleotide of four seed types in target polynucleotide With the determination of position.In some embodiments, sequence determination can (such as born of the same parents be phonetic by identifying the nucleotide of single type Pyridine) sequence in target polynucleotide " catcgc ... " and position, so that its sequence is represented as binary code, such as " 100101 ... " expression " c- (non-c) (non-c) c- (non-c)-c " etc. is realized.In some embodiments, these terms can be with The subsequence of the fingerprint for serving as the target polynucleotide including target polynucleotide；Uniquely identify one group of polynucleotides (such as All different RNA sequences expressed by cell) in a kind of target polynucleotide or a kind of target polynucleotide subsequence.

Present disclosure is not intended to be limited to the range of illustrated particular form, and is intended to cover variation described herein Alternative form, modification and the equivalent form of form.In addition, scope of the present disclosure completely include in view of present disclosure It may become other apparent versions to those skilled in the art.The scope of the present invention is only by appended claims To limit.

Sequence table

<110>Kun Ta bohr company

It ties up hereby the Metz Stefan C

Shi Diwenmengken

<120>using quencher based on optical nano-pore sequencing

<130> QNTPZ02000WO

<150> 62/377409

<151> 2016-08-19

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>random sequence

<400> 1

accgtttaaa ggtttccccg tcgta 25

Claims (22)

1. a kind of method of analysis of polynucleotide, which comprises

Make polynucleotides transposition by nano-pore, wherein different types of nucleotide generation of the polynucleotides is differentiable The different fluorescent labels of fluorescence signal, and wherein nucleotide is limited to successively move and passes through detection by the nano-pore Area；

Excite the fluorescent marker；

Use the fluorescence signal of the fluorescent marker of excitation of the non-fluorescent quencher quenching outside the detection zone；

When the fluorescent marker passes through the detection zone, the fluorescence signal emitted from the fluorescent marker is detected；And

The sequence of nucleotide is determined according to the fluorescence signal detected.

2. the method as described in claim 1, wherein the non-fluorescent quencher include be attached to its at least one is sudden Go out the oligonucleotides of part, and the wherein nano-pore only transposition single-chain nucleic acid, so that whenever described non-glimmering during transposition When optical trapping forces are in conjunction with the polynucleotides, the non-fluorescent quencher is removed from the polynucleotides.

3. method according to claim 2, wherein the nano-pore has outlet, and the detection zone includes neighbouring described Export and surround the volume of the outlet.

4. method as claimed in claim 3, wherein the polynucleotides have easy bit rate, and the detection zone is described The concentration of volume non-fluorescent quencher as described in the easy bit rate of the polynucleotides and the exit determines.

5. the method as described in claim 1, wherein the non-fluorescent quencher includes the intercalator of double-strand, and wherein described Nano-pore only transposition single-chain nucleic acid, so that during transposition when the non-fluorescent quencher is embedded in the polynucleotides, institute Non-fluorescent quencher is stated to be removed from the polynucleotides.

6. a kind of method of analysis of polynucleotide, which comprises

Polynucleotides transposition is set to pass through the nano-pore with outlet, wherein different types of nucleotide of the polynucleotides produces The different fluorescent labels of raw differentiable fluorescence signal, and wherein the nano-pore by the nucleotide be limited to according to Secondary movement passes through the detection zone in the nano-pore exit；

Excite the fluorescent marker；

The fluorescent marker of the excitation outside the detection zone is quenched using the non-fluorescent quencher in conjunction with the polynucleotides The fluorescence signal of object；

When the fluorescent marker passes through the detection zone, the fluorescence signal emitted from the fluorescent marker is detected；And

The sequence of nucleotide is determined according to the fluorescence signal detected.

7. method as claimed in claim 6, wherein the non-fluorescent quencher include be attached to its at least one is sudden Go out the oligonucleotides of part, and the wherein nano-pore only transposition single-chain nucleic acid, so that whenever described non-glimmering during transposition When optical trapping forces are in conjunction with the polynucleotides, the non-fluorescent quencher is removed from the polynucleotides.

8. the method for claim 7, wherein the detection zone includes the neighbouring outlet and the body for surrounding the outlet Product.

9. method according to claim 8, wherein the polynucleotides have easy bit rate, and the detection zone is described The concentration of volume non-fluorescent quencher as described in the easy bit rate of the polynucleotides and the exit determines.

10. a kind of method of the nucleotide sequence of determining polynucleotides, which comprises

(a) a kind of nanohole array is provided, the nanohole array includes with the first side, second side and what is passed through be more than The immobilon-p of one nano-pore, the immobilon-p separate the first Room and second Room, so that each nano-pore provides first Room Fluid communication between the second Room, and each nano-pore has the outlet for leading to the second Room and is customized ruler It is very little so that single-chain nucleic acid can not be passed through by double-strandednucleic acid, and each nano-pore limits the nucleotide of the polynucleotides of transposition It is made as successively moving the detection zone by its exit；

(b) make polynucleotides from first Room by the nano-pore transposition to the second Room, each polynucleotides have One or more fluorescent markers of its nucleotide are attached to, so that different types of nucleotide generates differentiable fluorescence The different fluorescent labels of signal；

(c) fluorescent marker is irradiated with excitation beam；

(d) fluorescent marker of the excitation outside the detection zone is quenched with the non-fluorescent quencher in conjunction with the polynucleotides The fluorescence signal of object；

(e) fluorescence signal of fluorescent marker of the detection in the detection zone；

(f) nucleotide sequence of the polynucleotides is determined according to the fluorescence signal detected.

11. method as claimed in claim 10, wherein the immobilon-p includes opaque layer, the opaque layer substantially hinders The transmission of the excitation beam is kept off, and wherein the illumination and the step of detection include falling and penetrating lighting system.

12. method as claimed in claim 10, wherein the non-fluorescent quencher is covalent with bromine, iodine, nitro, azo group The single stranded DNA combination dye of modification.

13. method as claimed in claim 12, wherein the single stranded DNA combination dye is asymmetric cyanine dye.

14. method as claimed in claim 10, wherein the non-fluorescent quencher includes bound fraction and quencher moieties.

15. method as claimed in claim 14, wherein the bound fraction is oligonucleotides or its analog.

16. method as claimed in claim 15, wherein the quencher moieties are Black Hole Quencher^TMCompound, flower Green quencher compound, xanthene quencher or anthraquinone quencher compound.

17. method as claimed in claim 10, wherein the detection zone is by the easy bit rate of the polynucleotides and described The concentration of non-fluorescent quencher described in two Room determines.

18. method as claimed in claim 10, wherein first Room is cis- room, and the second Room is trans- room.

19. method as claimed in claim 10, wherein the immobilon-p includes opaque layer, and the wherein illumination step Lighting system implementation is penetrated by falling.

20. method as claimed in claim 10, wherein the nano-pore of the nanohole array respectively contains and is fixed on institute State the protein nano hole in the hole of immobilon-p.

21. the method as described in any one of claims 1 to 20, wherein at least two of the variety classes nucleotide use extremely Few two kinds of different fluorescent labels.

22. the method as described in any one of claim 1 to 21, wherein described the step of being detected and determined includes (i) when more When nucleotide passes through detection zone, the time series of the fluorescence signal from the fluorescent marker is detected；(ii) separation comes From the fluorescence signal of different types of nucleotide；And the time of isolated fluorescence signal of (iii) basis from polynucleotides Sequence determines the sequence of nucleotide.