CN109252224A - A kind of cycling probe and the sequencing library construction method based on cycling probe capture - Google Patents
A kind of cycling probe and the sequencing library construction method based on cycling probe capture Download PDFInfo
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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Abstract
A kind of cycling probe and the sequencing library construction method based on cycling probe capture, this method comprises: by cycling probe and target nucleic acids anneal, wherein cycling probe is linear nucleic acid probe, it includes the specific arm area of two ends and the universal sequence of centre, wherein specific arm area with target nucleic acids for hybridizing, two ends head and the tail of probe are opposite after hybridization, the specific arm area that universal sequence is used to connect two ends and the identification region as universal primer；Under the action of polymerase and ligase, makes 3 ' ends of cycling probe expand with target nucleic acids template and connect to form ring molecule with 5 ' ends；Exonuclease is added to digest linear nucleic acid molecule.The present invention solves the problem of that capture rate is low and with high costs using cycling probe solution primer pair limited amount, solves the problems, such as that multiplex amplification homogeneity is poor using universal primer amplification using amplification principle.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of cycling probe and based on cycling probe capture
Sequencing library construction method.
Background technique
As sequencing technologies develop, the second generation is sequenced (NGS) and grows rapidly, and against high-throughput feature, becomes and grinds
Study carefully the main tool of DNA and RNA.In NGS, the sequencing that people can simultaneously synchronize up to 100,000,000 nucleic acid sequences,
Flux in base reaches the order of magnitude of Gb, and (WGS), which is sequenced, also to whole gene group becomes more and more conventional.Although
It can technically accomplish WGS, but it is the factor for limiting its application that the time, which is sequenced, and cost is sequenced.By to specific interested
Region is captured, and the major part in result can be both occupied to avoid the sequence information being not required to, and data volume needed for reducing finally saves
Cost-saving, can also be improved the sequencing depth of specific region, to provide more valuable information.
Particular region of interest is captured, there are two types of existing mainstream technology schemes: NimbleGenSeqCap technical side
Case and Ampliseq technical solution.NimbleGenSeqCap technical solution, capture object are the library of added connector, are led to
The tape label linear probe for being 50-105bp to target area design length is crossed, is hybridized and target area segment is enriched with,
Then high-flux sequence is carried out.Whole flow process includes the library construction of old process, label probe hybridization, Beads enrichment, target
Library elution, the secondary amplification in target library.Ampliseq technical solution, expands target area between Jian Ku, with
Increase the detection to target area in sequencing result.Technology designs one or more pairs of primers for each target area, these
There is overlapping between the amplified production end of primer, the corresponding PCR product in each region is allowed to cover whole region.PCR it
Afterwards, conventional NGS is carried out to PCR and builds library and sequencing.Due to have passed through exponential amplification, the nucleic acid of target area is in total nucleic acid
The ratio accounted for greatly promotes, therefore detection that can be used for low frequency mutation etc. is applied.
There is the following in the shortcomings that NimbleGenSeqCap technical solution: (1) capture rate is low, and complete outer chip is averagely caught
Efficiency is obtained in 50%-60%, small chip capture rate is average 40%；(2) chip cost is high, and single reaction price is minimum
1200 yuan or more, in addition elution reagent, single test cost is greater than 1500 yuan；(3) test operation complexity is cumbersome, and stability is poor,
It is unfavorable for industrialization promotion；(4) it is not suitable for the capture of smaller area, the smaller capture effect in region is poorer.Ampliseq technical side
The shortcomings that case, is with the presence of the following: (1) multiple PCR technique Stable Defects, and different primers amplification efficiency differs greatly, cannot
Effectively reflect the difference of the area of a room of different targets；(2) fragmentation sample it is not suitable for, significantly to fragmentation sample sensitivity
It reduces；(3) complex region primer inefficiency is difficult to capture；(4) PCR amplification recurring number is high, and the PCR mistake of introducing can shadow
Ring low frequency detection.
Summary of the invention
The present invention provides a kind of cycling probe and the sequencing library construction method based on cycling probe capture, have efficiently,
Low cost, the advantage that homogeneity is good, simple and efficient.
According in a first aspect, provided in a kind of embodiment it is a kind of based on cycling probe capture sequencing library construction method,
Include:
By cycling probe and target nucleic acids anneal, wherein above-mentioned cycling probe is linear nucleic acid probe comprising two
The specific arm area of a end and intermediate universal sequence, wherein above-mentioned specificity arm area is used to hybridize with above-mentioned target nucleic acids,
Two ends head and the tail of probe are opposite after hybridization, and above-mentioned universal sequence is used to connect the specific arm area of two ends and as logical
With the identification region of primer；
Under the action of polymerase and ligase, make 3 ' ends of above-mentioned cycling probe with above-mentioned target nucleic acids template into
Row expands and connect to form ring molecule with 5 ' ends；With
Exonuclease is added to digest linear nucleic acid molecule.
Further, above-mentioned universal primer is the primer with sequence measuring joints；Above-mentioned exonuclease divides linear nucleic acid
After son digestion, above-mentioned universal primer is added and carries out PCR amplification.
Further, above-mentioned universal primer is the primer without sequence measuring joints；Above-mentioned exonuclease divides linear nucleic acid
After son digestion, above-mentioned universal primer is added and carries out PCR amplification；After above-mentioned PCR amplification, amplified production is connected into sequencing and is connect
Head.
Further, it after above-mentioned amplified production connects upper sequence measuring joints, is cyclized and prepares DNA nanometers by rolling-circle replication
Ball.
Further, after above-mentioned exonuclease digests linear nucleic acid molecule, DNA nanometers are prepared by rolling-circle replication
Ball.
Further, it after above-mentioned amplified production connects upper sequence measuring joints, is cyclized and prepares DNA nanometers by rolling-circle replication
Ball.
Further, the specific arm section length of two ends of above-mentioned cycling probe is respectively 15~30 bases.
Further, the universal sequence length of the centre of above-mentioned cycling probe is from 25 bases to hundreds of bases.
Further, the G/C content of above-mentioned cycling probe is 30~70%, preferably 40~60%.
It further, include one section of random sequence in the universal sequence of the centre of above-mentioned cycling probe.
Further, 5 ' ends of above-mentioned universal primer include that different restriction enzyme sites is connect with adapting to different microarray datasets
Head.
According to second aspect, a kind of cycling probe for sequencing library building is provided in a kind of embodiment, is used for and target
Nucleic acids anneal hybridization is marked, above-mentioned cycling probe is linear nucleic acid probe comprising the specific arm area of two ends and centre
Universal sequence, wherein above-mentioned specificity arm area is used to hybridize with above-mentioned target nucleic acids, two end head and the tail phases of probe after hybridization
To specific arm area of the above-mentioned universal sequence for connecting two ends and the identification region as universal primer.
Method of the invention, using amplification principle, solve the problems such as capture rate is low and with high costs, individually react at
This is about 100-200 member, solves the problems, such as primer pair limited amount using cycling probe, is solved using universal primer amplification
The problem of multiplex amplification homogeneity difference, this method is simple and quick, can the interior completion at 8 from nucleic acid extraction to upper machine.
Detailed description of the invention
Fig. 1 is the principle signal of the sequencing library construction method based on cycling probe capture in one embodiment of the invention
Figure；
Fig. 2 is the principle signal of the sequencing library construction method based on cycling probe capture in another embodiment of the present invention
Figure；
Fig. 3 is the principle signal of the sequencing library construction method based on cycling probe capture in another embodiment of the invention
Figure；
Fig. 4 is the principle signal of the sequencing library construction method based on cycling probe capture in further embodiment of the present invention
Figure；
Fig. 5 is using the sequencing library construction method of the invention based on cycling probe capture and Ampliseq technology difference
Build BGISEQ-100 platform sequencing result comparison diagram behind library, wherein ordinate is to compare upper this kind of pathogenic bacteria gene group of database
Read (reads) quantity, Ec (black column), Ef (grey column), Ab (white column) respectively represent Escherichia coli, enterococcus faecium,
The case where Acinetobacter bauamnnii.
Specific embodiment
Below by specific embodiment combination attached drawing, invention is further described in detail.In the following embodiments and the accompanying drawings
In, many datail descriptions are in order to enable the present invention can be better understood.However, those skilled in the art can be without lifting an eyebrow
Recognize, part of feature is dispensed in varied situations, or can be by other elements, material, method institute
Substitution.In some cases, the relevant some operations of the present invention there is no display in the description or describe, this is to keep away
Exempt from core of the invention part to be flooded by excessive description, and to those skilled in the art, these phases are described in detail
It closes operation not to be necessary, they can completely understand according to the general technology knowledge of description and this field in specification
Relevant operation.
In the present invention, " cycling probe " is linear nucleic acid probe, and two ends of probe are that specific arm area is (such as long
Degree is 15~30 bases), specific arm area can hybridize with target nucleic acids, and the end head and the tail of probe are opposite after hybridization, therefore
Referred to as " cycling probe ".Centre be universal sequence, length for example from 25 bases to hundreds of bases (such as 100,200,
300,500,800 bases etc.), it can be become according to distance of the specific arm area of two ends in target nucleic acids
Change, the effect of universal sequence is the specific arm area for connecting two ends and the identification region as universal primer.One
In set system, the universal sequence of different probe identifies a pair of of universal primer jointly, therefore subsequent operation can be logical by a pair
The amplification of different cycling probes is completed with primer.The design of specific arm region sequence is similar with PCR primer, due to being directed to different targets
It is different to mark property between the arm area of nucleic acid, annealing needs DEG C gradient decline temperature from 98 DEG C to 56 to carry out.
In embodiments of the present invention, the G/C content of cycling probe is 30~70%, preferably 40~60%.Preferably implementing
It include one section of random sequence in the universal sequence of the centre of cycling probe, for determining the area between different amplicons in example
It is not derived from the mistake or polymorphic nucleic acid that amplification introduces.In a preferred embodiment, 5 ' ends of universal primer include not
Same restriction enzyme site is to adapt to different microarray dataset connectors.
Relative to the capture technique of linear probe, cycling probe must both ends be involved in annealing and could effectively connect, increase
The specificity of reaction.Cycling probe, can since two ends are connected by universal sequence compared with the beneficiation technologies of based on PCR
To avoid non-specific amplification caused by intersection between the latter's difference target primer.
The principle that Fig. 1 shows the sequencing library construction method based on cycling probe capture in one embodiment of the invention is shown
It is intended to.Design is directed to the specific cycling probe of targeting regions, and the intermediate ring region (black) of every probe is universal sequence, 5 ' ends
End and 3 ' ends be for target nucleic acids specific arm area, with have target sequence DNA anneal, in polymerase and company
Under the action of connecing enzyme, 3 ' ends of probe are connect using target sequence as template amplification and with 5 ' ends, and probe constitutes ring molecule.
Exonuclease is added to digest linear DNA molecule, adds the primer with sequence measuring joints and carries out PCR amplification, amplified production
It can be used to BGISEQ-100 sequencing after purified.
Fig. 2 shows the principles of the sequencing library construction method based on cycling probe capture in another embodiment of the present invention
Schematic diagram.Design is directed to the specific cycling probe of targeting regions, and the intermediate ring region (black) of every probe is universal sequence, and 5 '
End and 3 ' ends are the specific arm area for target nucleic acids, and the DNA anneal for having target sequence, in polymerase and
Under the action of ligase, 3 ' ends of probe are connect using target sequence as template amplification and with 5 ' ends, and probe constitutes cyclic annular point
Son.Exonuclease is added to digest linear DNA molecule, the universal primer added for ring region carries out PCR amplification.Later will
Amplified production adds sequence measuring joints, can be used to BGISEQ-100 sequencing.
Fig. 3 shows the principle of the sequencing library construction method based on cycling probe capture in another embodiment of the invention
Schematic diagram.Design is directed to the specific cycling probe of targeting regions, and the intermediate ring region (black) of every probe is universal sequence, and 5 '
End and 3 ' ends are the specific arm area for target nucleic acids, and the DNA anneal for having target sequence, in polymerase and
Under the action of ligase, 3 ' ends of probe are connect using target sequence as template amplification and with 5 ' ends, and probe constitutes cyclic annular point
Son.Exonuclease is added to digest linear DNA molecule, the universal primer added for ring region carries out PCR amplification.Later will
Amplified production connects top connection and then prepares DNA nanosphere (DNB), directly by cyclisation (such as by split oligo)
The upper machine sequencing of BGISEQ-500.
Fig. 4 shows the principle of the sequencing library construction method based on cycling probe capture in further embodiment of the present invention
Schematic diagram.Design is directed to the specific cycling probe of targeting regions, and the intermediate ring region (black) of every probe is universal sequence, and 5 '
End and 3 ' ends are the specific arm area for target nucleic acids, and the DNA anneal for having target sequence, in polymerase and
Under the action of ligase, 3 ' ends of probe are connect using target sequence as template amplification and with 5 ' ends, and probe constitutes cyclic annular point
Son.Exonuclease is added to digest linear DNA molecule.Then it prepares DNA nanosphere (DNB), machine on direct BGISEQ-500
Sequencing.
It should be noted that above-mentioned example embodiment mainly lists current more common application platform range, it should
Technology is the general beneficiation technologies of genome, can be used for other two generations or three generations's microarray dataset, such as Proton, Pacbio
With Qiagen microarray dataset etc..
The technical solution that the present invention will be described in detail by the following examples, it should be understood that embodiment is merely exemplary, no
It can be interpreted as limiting the scope of the invention.
Embodiment 1: the Analysis of pathogenic bacteria based on BGISEQ-100 microarray dataset
Sequencing library structure is carried out to sample using the cycling probe technology of the embodiment of the present invention and Ampliseq technology respectively
It builds, the library of two kinds of technologies is using different bar codes to distinguish.
In the cycling probe technology of the embodiment of the present invention, searching Escherichia coli (Ec), enterococcus faecium (Ef), Bao Man are motionless
A cycling probe (nucleic acid is designed for internal sequence in the conservative region of 3 500bp in bacillus (Ab) genome, each region
Probe and primer are synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd), probe sequence is as shown in table 1:
Table 1
Escherichia coli (Ec), enterococcus faecium (Ef), the bacterium solution of Acinetobacter bauamnnii (Ab) are extracted into nucleic acid (using day respectively
Root DP-316 kit), the nucleic acid of extraction is carried out using Qubit dsDNA HS Assay Kit quantitatively and according to respective gene
Group size calculates the cell concentration before extracting, and is diluted to nucleic acid is equivalent to 1000 copies/mL concentration respectively, then by three seed nucleus
Acid is mixed by volume equal proportion.
The reagent information that the present embodiment uses is as shown in table 2 below:
Table 2
1, probe anneals
Cycling probe (Ec_1~Ab_3) is mixed with same molar ratio, is added in the genome DNA sample of extraction, shape
At reaction system as shown in table 3 below, be heated to 98 DEG C gradually gradient cooling kept for 2 hours to 56 DEG C, and at 56 DEG C, make DNA
Double-strand in sample is opened and is gradually hybridized with cycling probe.Table 3 shows the reaction system and program of probe anneals:
Table 3
2, notch filling-in
After the completion of anneal, reaction system as shown in table 4 below is configured, and the reaction system is added to probe anneals
In reaction system afterwards, mix, reacted according to response procedures shown in table 4, make probe 3 ' ends extend and with 5 ' end
End connection, circularizing probes.
Table 4
3, circumscribed enzymic digestion
1 μ L Exo I (20U/ μ L), 1 μ L Exo III (100U/ μ L) are added into product, according to as shown in table 5 below
Response procedures are reacted, the linear nucleic acid in digestion system, and 80 DEG C of incubations inactivate excision enzyme in 10 minutes later.
Table 5
4, PCR amplification
PCR reaction system as shown in table 6 below is configured, and is reacted according to response procedures shown in table 6, to the probe of cyclisation
Carry out PCR amplification.
Table 6
5, it purifies
PCR product is purified by Axygen magnetic bead, obtains library.Specifically comprise the following steps:
(1) PCR product is supplied with water to 100 μ L and 80 μ L (0.8 times) Axygen magnetic bead is added, mix well rear room temperature
Stand 5min；
(2) by previous step solution --- the of short duration centrifugation of magnetic bead mix is placed on 2min on magnetic frame, carefully turns supernatant
Move on to new 1.5ml EP pipe；
(3) 20 μ L (0.2 times of original volume) Axygen magnetic bead is added into the supernatant of previous step, it is quiet to mix well rear room temperature
5min is set, of short duration centrifugation is placed on 2min on magnetic frame, carefully discards supernatant；
(4) it is carefully added into 500 μ L, 80% ethyl alcohol, and (number of revolutions is generally 2 to rotating centrifugal pipe sufficiently to wash magnetic bead
It is secondary), static 1min after washing inhales and abandons ethyl alcohol；
(5) it is primary that step (4) are repeated；
(6) room temperature dries (length of time is related with indoor humidity, generally 5min) after ethyl alcohol is abandoned in careful suction, until magnetic bead
Matte surface；
(7) it is added 20 μ L EB solution (repeatedly careful piping and druming mixes magnetic bead), static 5min (flicking tube wall mixing in interval)；
(8) of short duration centrifugation (time that time slightly long magnetic bead dries may be shorter) is placed on 2min on magnetic frame, careful to inhale
Take solution into new 1.5mL centrifuge tube.
6, upper machine sequencing
The DNA library of purifying is diluted to suitable concentration, the library with the building of Ampliseq technology is according to equimolar concentration
Mixing, with machine sequencing analysis on BGISEQ-100 platform.
The genome alignment of lower machine data and three kinds of pathogens will be only capable of counting than a kind of upper read of cause of disease, unite
The read situation (referring to Fig. 5) of three kinds of pathogens in different technologies is counted, cycling probe technology builds the sequencing result in library as the result is shown
Difference between middle various pathogenic bacteria is smaller.However, the bacterium of three kinds of same concentrations exists in the result that Ampliseq technology builds library
It is certain poor to illustrate that Ampliseq technology builds primer pair amplifies efficiency presence in library for differing greatly between data volume in sequencing result
It is different, lead to the generation for expanding inhomogeneity.
Embodiment 2: the Analysis of pathogenic bacteria based on BGISEQ-500 microarray dataset
In the embodiment of the present invention, based on the technical principle of BGISEQ-500 platform, text can be removed in the library construction stage
Library PCR step shortens the time of Analysis of pathogenic bacteria, obtains analysis result more quickly.
In the cycling probe technology of the embodiment of the present invention, Escherichia coli (Ec), enterococcus faecium (Ef), Acinetobacter bauamnnii
(Ab) conservative region being directed in genome is same as Example 1, and a cycling probe is designed for internal sequence in each region,
Each probe sequence designs one group of probe primer (F, R).Universal sequence (Linker) and every group of probe in the middle part of probe draw
Object is synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd), nucleic acid sequence is as shown in table 7:
Table 7
Escherichia coli (Ec), enterococcus faecium (Ef), the bacterium solution of Acinetobacter bauamnnii (Ab) are extracted into nucleic acid (using day respectively
Root DP-316 kit), the nucleic acid of extraction is carried out using Qubit dsDNA HS Assay Kit quantitatively and according to respective gene
Group size calculates the cell concentration before extracting, and is diluted to nucleic acid is equivalent to 1000 copies/mL concentration respectively, then by three seed nucleus
Acid is mixed by volume equal proportion.
The reagent information that the present embodiment uses is as shown in table 8 below:
Table 8
| Reagent | Producer | Article No. |
| Taq DNA Ligase | NEB | M0208S |
| dNTP | Enzymatics | N205L |
| Phusion | Thermo Fisher | F-530L |
| Exo I | NEB | M0293S |
| Exo III | NEB | M0206S |
| 2×Phusion Master Mix | Thermo Fisher | F-531 |
1, probe PCR synthesizes
PCR reaction system as shown in table 9 below is configured, and is reacted according to response procedures shown in table 9, Linker is carried out
PCR amplification.9 conservative regions are directed in the embodiment of the present invention, it is therefore desirable to PCR synthesis be carried out to the probe in 9 regions respectively.
Table 9
2, cycling probe purifies
PCR product is purified by Axygen magnetic bead, obtains cycling probe.Specifically comprise the following steps:
(1) PCR product is supplied with water to 100 μ L and 100 μ L Axygen magnetic beads is added, be stored at room temperature after mixing well
5min；
(2) by previous step solution --- the of short duration centrifugation of magnetic bead mix is placed on 2min on magnetic frame, carefully turns supernatant
Move on to new 1.5ml EP pipe；
(3) it is carefully added into 500 μ L, 80% ethyl alcohol, and (number of revolutions is generally 2 to rotating centrifugal pipe sufficiently to wash magnetic bead
It is secondary), static 1min after washing inhales and abandons ethyl alcohol；
(4) it is primary that step (4) are repeated；
(5) room temperature dries (length of time is related with indoor humidity, generally 5min) after ethyl alcohol is abandoned in careful suction, until magnetic bead
Matte surface；
(6) it is added 20 μ L EB solution (repeatedly careful piping and druming mixes magnetic bead), static 5min (flicking tube wall mixing in interval)；
(7) of short duration centrifugation (time that time slightly long magnetic bead dries may be shorter) is placed on 2min on magnetic frame, careful to inhale
Take solution into new 1.5mL centrifuge tube.
The Qubit dsDNA HS Assay Kit kit quantification of probe after purification, is mixed into 1 μ for different probes
The probe liquid of every kind of M/, -20 DEG C store for future use, and probe liquid can prepare in advance and use for multiple library construction, without in text
It is just prepared when library constructs.
3, probe anneals
According to table 10 prepare reaction system, be heated to 98 DEG C gradually gradient cooling to 56 DEG C, and 56 DEG C keep 2 hours,
It opens the double-strand in DNA sample and gradually hybridizes with cycling probe.Table 10 shows the reaction system and program of probe anneals:
Table 10
4, notch filling-in
After the completion of anneal, reaction system as shown in table 11 below is configured, and the reaction system is added to probe and is moved back
It in reaction system after fire, mixes, is reacted according to response procedures shown in table 11, extend 3 ' ends of probe and with 5 '
End connection, circularizing probes.
Table 11
5, circumscribed enzymic digestion
1 μ L Exo I (20U/ μ L), 1 μ L Exo III (100U/ μ L) are added into product, according to as shown in table 12 below
Response procedures are reacted, the linear nucleic acid in digestion system, and 80 DEG C of incubations inactivate excision enzyme in 10 minutes later.
Table 12
6, it purifies
Digestion product is purified by Axygen magnetic bead, obtains cyclic annular library.Specifically comprise the following steps:
(1) digestion product is supplied with water to 50 μ L and 50 μ L Axygen magnetic beads is added, be stored at room temperature after mixing well
5min；
(2) by previous step solution --- the of short duration centrifugation of magnetic bead mix is placed on 2min on magnetic frame, carefully turns supernatant
Move on to new 1.5ml EP pipe；
(3) it is carefully added into 500 μ L, 80% ethyl alcohol, and (number of revolutions is generally 2 to rotating centrifugal pipe sufficiently to wash magnetic bead
It is secondary), static 1min after washing inhales and abandons ethyl alcohol；
(4) it is primary that step (3) are repeated；
(5) room temperature dries (length of time is related with indoor humidity, generally 5min) after ethyl alcohol is abandoned in careful suction, until magnetic bead
Matte surface；
(6) it is added 20 μ L EB solution (repeatedly careful piping and druming mixes magnetic bead), static 5min (flicking tube wall mixing in interval)；
(7) of short duration centrifugation (time that time slightly long magnetic bead dries may be shorter) is placed on 2min on magnetic frame, careful to inhale
Take solution into new 1.5mL centrifuge tube.
6, upper machine sequencing
The cyclic annular library of purifying is diluted to suitable concentration, DNB is prepared according to the requirement of BGISEQ-500 platform, upper machine is surveyed
Sequence analysis.
Use above specific case is illustrated the present invention, is merely used to help understand the present invention, not to limit
The system present invention.For those skilled in the art, according to the thought of the present invention, can also make several simple
It deduces, deform or replaces.
SEQUENCE LISTING
<110>Shenzhen Hua Da gene limited liability company
<120>a kind of cycling probe and the sequencing library construction method based on cycling probe capture
<130> 17I24630
<160> 30
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<213>artificial sequence
<400> 17
cctgagcaac ttcgtatttg ggccattaca gtcggaggcc aagcgg 46
<210> 18
<211> 33
<212> DNA
<213>artificial sequence
<400> 18
tattgttgtg ttttgtagtc ggatcgtagc cat 33
<210> 19
<211> 43
<212> DNA
<213>artificial sequence
<400> 19
gtcgtggaat tcgtgttgca taaacgagtc ggaggccaag cgg 43
<210> 20
<211> 36
<212> DNA
<213>artificial sequence
<400> 20
aagcatcaaa cacaacagca gtcggatcgt agccat 36
<210> 21
<211> 45
<212> DNA
<213>artificial sequence
<400> 21
cagtagaccc aatcagtaaa atcagcagag tcggaggcca agcgg 45
<210> 22
<211> 33
<212> DNA
<213>artificial sequence
<400> 22
ctgggtattt gtttttagtc ggatcgtagc cat 33
<210> 23
<211> 46
<212> DNA
<213>artificial sequence
<400> 23
cgaaatacat ggtcttggca tccaacttca gtcggaggcc aagcgg 46
<210> 24
<211> 33
<212> DNA
<213>artificial sequence
<400> 24
ttttggtcag gtgcatagtc ggatcgtagc cat 33
<210> 25
<211> 46
<212> DNA
<213>artificial sequence
<400> 25
gtacgacttc agtggcgatg tgtatcgcaa gtcggaggcc aagcgg 46
<210> 26
<211> 33
<212> DNA
<213>artificial sequence
<400> 26
ttccaatcgc ttctctagtc ggatcgtagc cat 33
<210> 27
<211> 42
<212> DNA
<213>artificial sequence
<400> 27
gtttcagggg cgttgtagtg tccgtagtcg gaggccaagc gg 42
<210> 28
<211> 35
<212> DNA
<213>artificial sequence
<400> 28
attaccaatc gctgcaccag tcggatcgta gccat 35
<210> 29
<211> 44
<212> DNA
<213>artificial sequence
<400> 29
gcagcaacag catctagtta ctcaagcagt cggaggccaa gcgg 44
<210> 30
<211> 34
<212> DNA
<213>artificial sequence
<400> 30
aaaacacaat gaaccagagt cggatcgtag ccat 34
Claims (10)
1. a kind of sequencing library construction method based on cycling probe capture, which is characterized in that the described method includes:
By cycling probe and target nucleic acids anneal, wherein the cycling probe is linear nucleic acid probe comprising two ends
The specific arm area at end and intermediate universal sequence, wherein the specificity arm area hybridizes for hybridizing with the target nucleic acids
Two ends head and the tail of probe are opposite afterwards, and the universal sequence is used to connect the specific arm area of two ends and draws as general
The identification region of object；
Under the action of polymerase and ligase, expand 3 ' ends of the cycling probe with the target nucleic acids template
Increase and connect to form ring molecule with 5 ' ends；With
Exonuclease is added to digest linear nucleic acid molecule.
2. sequencing library construction method according to claim 1, which is characterized in that the universal primer is connect with sequencing
The primer of head；After the exonuclease digests linear nucleic acid molecule, the universal primer is added and carries out PCR amplification.
3. sequencing library construction method according to claim 1, which is characterized in that the universal primer is connect without sequencing
The primer of head；After the exonuclease digests linear nucleic acid molecule, the universal primer is added and carries out PCR amplification；Institute
After stating PCR amplification, amplified production is connected into upper sequence measuring joints；
Preferably, it after the amplified production connects upper sequence measuring joints, is cyclized and DNA nanosphere is prepared by rolling-circle replication.
4. sequencing library construction method according to claim 1, which is characterized in that the exonuclease is by linear nucleic acid
After molecule digestion, DNA nanosphere is prepared by rolling-circle replication.
5. sequencing library construction method according to any one of claims 1 to 4, which is characterized in that the cycling probe
The specific arm section length of two ends is respectively 15 ~ 30 bases.
6. sequencing library construction method according to any one of claims 1 to 4, which is characterized in that the cycling probe
Intermediate universal sequence length is from 25 bases to hundreds of bases.
7. sequencing library construction method according to any one of claims 1 to 4, which is characterized in that the cycling probe
G/C content be 30 ~ 70%, preferably 40 ~ 60%.
8. sequencing library construction method according to any one of claims 1 to 4, which is characterized in that the cycling probe
It include one section of random sequence in intermediate universal sequence.
9. sequencing library construction method according to any one of claims 1 to 4, which is characterized in that the universal primer
5 ' ends include different restriction enzyme sites to adapt to different microarray dataset connectors.
10. a kind of cycling probe for sequencing library building, which is characterized in that the cycling probe with target nucleic acids for moving back
Fire hybridization, the cycling probe are linear nucleic acid probe comprising the specific arm area of two ends and intermediate universal sequence,
Wherein for hybridizing with the target nucleic acids, two ends head and the tail of probe are opposite after hybridization, described logical in the specific arm area
Use specific arm area of the sequence for connecting two ends and the identification region as universal primer.
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| CN110029147A (en) * | 2019-01-25 | 2019-07-19 | 上海何因生物科技有限公司 | A kind of single tube realizes the non-specific PCR amplification method of continuum |
| CN112063690A (en) * | 2020-09-18 | 2020-12-11 | 北京求臻医学检验实验室有限公司 | Construction method and application of single-molecule probe multi-target capture library |
| CN112266948A (en) * | 2020-11-06 | 2021-01-26 | 中山大学孙逸仙纪念医院 | High-throughput targeting library building method and application |
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| CN110029147A (en) * | 2019-01-25 | 2019-07-19 | 上海何因生物科技有限公司 | A kind of single tube realizes the non-specific PCR amplification method of continuum |
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