EP1234182A2 - Hybridisierung von ziel-dna mit immobilisierten nukleinsäureanaloga - Google Patents
Hybridisierung von ziel-dna mit immobilisierten nukleinsäureanalogaInfo
- Publication number
- EP1234182A2 EP1234182A2 EP00948560A EP00948560A EP1234182A2 EP 1234182 A2 EP1234182 A2 EP 1234182A2 EP 00948560 A EP00948560 A EP 00948560A EP 00948560 A EP00948560 A EP 00948560A EP 1234182 A2 EP1234182 A2 EP 1234182A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- group
- dna
- soft
- soft metal
- hybridization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
- C07K14/003—Peptide-nucleic acids (PNAs)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
- B01J20/0225—Compounds of Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
- B01J20/0233—Compounds of Cu, Ag, Au
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
- B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
- B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
- B01J20/3297—Coatings in the shape of a sheet
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
Definitions
- This invention relates to the use of peptide nucleic acids and analogs in hybridization reactions, particularly on microarrays.
- DNA and ribonucleic acid are macromolecules built up from simple monomeric subunits called nucleotides.
- the nucleotide has the following three components: 1) a cyclic five-carbon (pentose) sugar (deoxyribose for DNA, and ribose for RNA), 2) a nitrogenous base of either purine or pyrimidine derivation, covalently attached to the l'-carbon atom of the sugar by a N-glycosylic bond, and 3) a phosphate attached to the 5' carbon of the sugar by a phosphodiester bond.
- the purines are adenine (A) and guanine (G), while the pyrimidines are cytosine (C) and thymine (T) for DNA, and cytosine (c) and uracil (U) for RNA.
- nucleotides of DNA are called deoxyribonucleotides, whereas those of RNA are called ribonucleo tides.
- Each nucleotide contains both a specific and a nonspecific region.
- the phosphate and sugar groups are the nonspecific portions of the nucleotide, while the purine and pyrimidine bases make up the specific portion.
- Nucleotides are joined to one another linearly by a chemical bond between atoms in the nonspecific regions to form polynucleotides. The linkage, called a phosphodiester bond, is between a phosphate group and a hydroxyl group on the sugar component.
- DNA usually consists of two complementary strands coiled about one another to form a double helix. There are ten nucleotides on each chain for every turn of the helix. The two chains are joined together through a combination of van der Waals forces and hydrogen bonds between the purine and pyrimidine base pairs on complementary strands. This base pairing is so specific that adenine binds only to thymine and guanine only to cytosine. This base pairing provides stabilization by hydrogen bonding between complementary bases.
- this specificity of base pairing is what permits the transmission of genetic information from one generation to another.
- DNA double helix unwinds, and two new complementary DNA strands are formed.
- the sequence of bases (A, G, T, and C) in a strand of DNA specifies which amino acids are assembled in what order to form proteins.
- Each amino acid is encoded by a three base sequence of nucleotides, known as a codon; the correlation between each amino acid and its codon is known as the genetic code.
- the code is degenerate in that several different codons encode the same amino acid.
- PNA peptide nucleic acid
- nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences.
- the peptide nucleic acids as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
- PNAs are compounds that are analogous to oligonucleotides, but differ in composition.
- PNAs the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone.
- Each subunit of the peptide backbone is attached to a naturally- occurring or non-naturally-occurring nucleobase.
- One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
- PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes.
- PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (T m ).
- T m melting temperatures
- This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes.
- Another advantage of PNA/DN A(RNA) duplexes is that T m is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
- Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA) 2 /DNA(RNA) triplexes of high thermal stability (Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc, 1992, 114, 1895; Egholm et al., J. Am. Chem. Soc, 1992, 114, 9677).
- PNAs In addition to increased affinity, PNAs have increased specificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8° to 20° C. drop in the T m relative to the DNA/DNA duplex. This decrease in T m is not observed with the corresponding DNA/DNA duplex mismatch (Egholm et al., Nature 1993, 365, 566).
- a further advantage of PNAs compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
- PNAs over DNA (or RNA) for use as in situ hybridization probes are their: (1) higher thermal stability, (2) higher specificity, and (3) resistance to degrading enzymes.
- the neutral backbone of the PNAs gives to PNA:DNA duplexes (or PNA: RNA) a higher thermal stability compared to DNA: DNA (or RNA:RNA) duplexes. This stronger binding is attributed to the lack of charge repulsion between the PNA strand and the DNA (or RNA) strand.
- PNAs show a greater specificity in binding to complementary DNA (or RNA). As a result a PNA/DNA mismatch is more destabilizing than a mismatch in a DNA/DNA duplex.
- PNA oligomers are also resistant to degradation by proteases and nucleases since their polyamide backbone with nucleobase side chains is not a combination easily recognized by these enzymes. This extends the lifetime of any system or device in which PNAs are used.
- the object of the present invention is to provide a method for the immobilization of labeled PNAs onto solid surfaces for use in hybridization, purification, biosensing, and other biochemical applications.
- This invention is related to the immobilization of peptide nucleic acids (PNAs) onto solid surfaces for use in hybridization, purification, biosensing, and other biochemical applications. Specifically, their use to increase the thermal stability, specificity, and lifetime of devices based on in situ hybridization is disclosed. A method is disclosed by which peptide nucleic acids replace oligonucleotides as probes in the detection of specific DNA and RNA sequences.
- PNAs peptide nucleic acids
- the general process involves the use of solid substrates containing specific functionalities to which the PNAs are immobilized.
- the solid supports are activated by sorbing the following construct in the order given: (1) an anchor group; (2) a spacer arm; and (3) a reactive terminal group.
- This tripartite structure provides a stable anchor bond to the solid support, a spacer arm which gives flexibility to the PNA allowing it to interact with its environment in a way which minimizes any steric hindrance, and a reactive terminal group to immobilize the PNA.
- This invention provides a ligand-binding solid support having a soft metal solid surface and a heterobifunctional spacer chemi- or physisorbed to the soft metal solid surface via soft metal-soft base bonding.
- the soft metal solid surface is silver, copper, gold, platinum (II), mercury, mercury (II), thallium, cadmium (II), platinum (IV) or palladium (II).
- the heterobifunctional spacer is preferably a hydrocarbon of chain length from about 10 to about 40 carbon atoms, having at least one soft base anchor group and at least one nucleotide binding group.
- the soft base anchor group is an RSH, RS ⁇ R 2 S, RSSR, CN " , S 2 O 3 2 -, I " , R 3 P, (RO) 3 P, C2H4 or C6H6 group, where R is an organic group.
- an oligonucleotide is pre-attached to the spacer.
- This invention also provides methods for preparing a ligand-binding solid surface, by selecting a soft metal solid surface and immobilizing a heterobifunctional spacer on said solid surface via soft metal-soft base bonding.
- Assay systems having soft metal solid surfaces and a heterobifunctional spacer chemi- or physisorbed to said soft metal solid surface via soft metal-soft base bonding are also provided, as are methods for detecting the presence of a biological molecule by exposing a sample containing biological molecules to a surface as defined above.
- Anchor group refers to the functional chemical group containing the soft base that sorbs the spacer to the soft metal surface.
- Binding density refers to the number of reactive terminal groups per unit surface area available for binding the labeled biopolymer.
- Biopolymer refers to biological molecules such as proteins, oligonucleotides, DNA, RNA, PNA, etc., which are the basis of hybridization, purification, immunoassays, and many other biochemical applications.
- Hybridization refers to binding reaction between complementary partners of biopolymer molecules.
- Ligand refers to one member of the ligand/receptor binding pair, such as, oligonucleotides, DNA, and proteins.
- Nonspecific interaction refers to the individual physico-chemical interactions (i.e., hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals forces) where structure is not involved.
- Protein refers to enzymes, antibodies, and any other polypeptides.
- Soft bases refer to the species defined as having a small charge and large size and preferring to bind with soft metals.
- Soft metals refer to the species defined as having a small charge and large size and preferring to bind with soft bases.
- Specific interactions refers to the sum total of a particular set of physico- chemical interactions where structure can play a major role. These interactions include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals forces. “Steric hindrance” refers to the effect by large groups near the ligand, which limits its accessibility to the receptor molecule. Immobilization of molecules on so t metal surfaces
- the general process involves the use of substrates containing soft metal thin films .
- Heterobifunctional spacer molecules are then added.
- This heterobifunctional spacer is a hydrocarbon having a chain length of about 10 to about 40 carbon atoms, preferably about 15 to about 25 carbon atoms, having at least two functional groups. Of the two functional groups, one is a soft base that will sorb with the soft metal surface.
- the other functional group on the spacer is selected to bind the functional group on the label of the ligand.
- an oligonucleotide is pre-attached to the spacer prior to sorption on the metal surface. This process creates an active solid surface that is able to bind labeled ligands in high density and with minimum nonspecific binding.
- the soft base anchor group is an RSH, RS ⁇ R 2 S, RSSR, CN ⁇ S 2 O 3 2 ⁇ I ⁇ R 3 P, (RO) 3 P, C2H4 or C6H6 group, where R is an organic group.
- the present invention also provides methods for recovering the immobilized ligands by using sulfur-containing competing molecules to displace the heterobifunctional spacers. Due to the high aqueous solubility of thiodiglycol and its thioether functional group, a high elution recovery can be accomplished using a concentrated solution of thiodiglycol. The substrate may then be reused by washing with water and ethanol followed by heating under a partial vacuum in order to drive off the relatively volatile thiodiglycol. The sorbed molecules are bound to the solid surface by valence forces similar in strength to those involved in covalent bonds. However, unlike covalent interactions, there is a dynamic equilibrium in which adsorbed molecules can be desorbed without breaking any bonds. The interaction between soft metal ions and soft bases is described qualitatively by the principle of Hard and Soft Acids and Bases (HSAB) based on the Lewis definition of acids and bases (Pearson, R. G., Chem. Brit. 1967, 3, 103-107.
- HSAB Hard and Soft Acids and Base
- Van der Waals dispersion energies between atoms or groups in the same molecule may lead to the stabilization of the molecule. These forces are large in complexes formed by highly polarizable soft acids and bases, thus providing additional stability.
- the first step in the immobilization process is the fabrication of soft metal thin films (about 20nm) on the substrate of choice (e.g., fused silica, lime glass, quartz, oxidized silicon, etc.). This is done by well known methods such as electron beam evaporation.
- the substrate of choice e.g., fused silica, lime glass, quartz, oxidized silicon, etc.
- heterobifunctional spacer arm After washing and drying, the heterobifunctional spacer arm is absorbed.
- Various types of heterobifunctional spacers are commercially available or protocols for their synthesis can be found in the literature.
- the spacer At least one is a soft base to bind the soft metal surface.
- One other functional group is reactive towards the ligands or biomolecules to be immobilized. All of these chemical groups and reactions are well known to those skilled in the art and some examples are shown in TABLE 2.
- the functional groups can depend on the type of biomolecule to be immobilized. For example, all proteins contain an amino group on one end and a carboxylate group on the other end, besides all other functional groups provided by the specific amino acids on the sequence. In the case of oligonucleotides these are usually synthesized one nucleotide at a time. Because of this, a single nucleotide label with the desired functional group can be added at some point in the synthesis (usually at the beginning or the end), thus labeling the resulting oligonucleotide. These individual nucleotides can be modified either chemically or enzymatically with any type of functional group in order to provide the desired label.
- This chemical or enzymatic labeling can be extended to DNA molecules, with the difference that all bases within the molecule targeted by the labeling reaction will be modified. If the desired result is to label the DNA molecule only at one point, the best approach is polymerase chain reaction (PCR) amplification using primers that have been already modified with the desired functional group.
- PCR polymerase chain reaction
- the molecule After immobilizing the desired target molecule and performing the desired biochemical application, the molecule can be recovered and the surface regenerated. This can be done by a procedure known as elution.
- a very common mode of elution of specifically bound molecules is the use of competing molecules, which displace the bound molecule. In order to chose a proper displacer it is important to take into account the nature of the specific interaction.
- Ligands immobilized through soft metal/soft base interactions on soft metal thin films may be recovered through the use of sulfur- containing competing molecules which displace the heterobifunctional spacers.
- a high elution recovery can be accomplished using a concentrated solution of thiodiglycol.
- the substrate may then be reused.
- PNA solutions of a specific composition are delivered to specific sites on the solid surface.
- the functional groups on the solid surface react with the functional groups on the PNA to form a stable bond. This is repeated at different sites on the solid surface using PNA solutions of different compositions, yielding a heterogeneous surface divided into specific reaction sites.
- Each of these reaction sites contains PNAs, which are complementary to specific sequences of DNA or RNA.
- This solid surface with the immobilized probe PNA molecules is then contacted with labeled target DNA or RNA solutions.
- These target molecules are extracted from cells of unknown genetic composition and labeled with molecules (e.g., fluorophores, radioisotopes, etc) that are easily identified by regular detection methods.
- the identity of the genetic make-up of these cells is determined by hybridization to their complementary PNA probes of known composition. After the hybridization step, the solid surface is washed to remove any unbound DNA or RNA. The washed surface is then ready for the detection and quantification of the hybridized target by measuring the amount of label on each of the sites.
- Silicon chips with platinum thin films were manufactured by electron beam evaporation. Prior to use, these surfaces are cleansed by using a mixture of 13% RBS 35 solution (Pierce) and 33% ethanol in deionized water. The chips are washed in this solution by immersing in an ultrasonic bath at 50°C for 20 minutes. This is followed by rinsing three times in deionized water using an ultrasonic bath at 50°C for 10 minutes. After rinsing the chips are blow-dried under nitrogen or argon.
- the hetrobifunctional spacer arm was succinimidyl-6- (biotinamido)hexanoate.
- This molecule is commercially available (Pierce Chemical Co.) or can be synthesized using the information available in the literature (Staros, J. V., Biochemistry, 1982, 27( 7):3950-3955).
- This molecule is a derivative of D-biotin containing an 6-aminocaproic acid spacer arm, about 30.5 A in length, attached to the valeric acid side chain of biotin and terminating in an NHS ester.
- This NHS ester reacts with amine groups in proteins and other molecules to form stable amide bond derivatives.
- Optimal reaction conditions are at pH 7-9.
- Amine-containing buffers such as Trizma, which may compete in the acylation reaction should be avoided.
- This spacer arm molecule is insoluble in aqueous reaction conditions and must be dissolved in organic solvents prior to the addition to the aqueous buffered reaction solution.
- a stock solution may be prepared in either of the organic solvents N,N-dimethylformamide (DMF) or dimethylsulfoxide (DMSO). Addition to the aqueous solution should not exceed 10% organic solvent to avoid precipitation.
- the molar ratio of the spacer arm molecule to a protein should be 2-50:1 with higher levels resulting in higher incorporation yields.
- the activated chips are submerged in a 10 mg/ml solution of the amino-labeled oligonucleotide in 0.1 M sodium phosphate, 0.15 M NaCl, at a pH of 7.2 for 30-60 minutes at room temperature, or for several hours at 4°C.
- the chips are then washed three times in the phosphate buffer followed by drying under a stream of nitrogen.
- Silicon chips with gold thin films were manufactured by electron beam evaporation. Prior to use these surfaces are cleansed by using a mixture of 13% RBS 35 solution (Pierce) and 33% ethanol in deionized water. The chips are washed in this solution by immersing in an ultrasonic bath at 50°C for 20 minutes. This is followed by rinsing three times in deionized water using an ultrasonic bath at 50°C for 10 minutes. After rinsing the chips are blow-dried under nitrogen or argon. For this example the hetrobifunctional spacer arm was dithiobis(succinimidyl- undecanoate). This molecule can be synthesized using the information available in the literature (Wagner, et al, Biophys.
- the molecule is made up by two molecules each containing a dodecanethiol spacer arm attached to an NHS ester and held together through a disulfide bond.
- the activation of the soft metal surface has to be done in the presence of a disulfide reductant buffer such as dithiothreitol and dioxane. This breaks the disulfide bond and leads to two heterobifunctional crosslinkers with a NHS ester for binding amino-containing ligands and a thiol group attached to the soft metal surface.
- the NHS ester reacts with amine groups in proteins and other molecules to form stable amide bond derivatives.
- Optimal reaction conditions are at pH 7-9.
- Amine-containing buffers such as Trizma, which may compete in the acylation reaction should be avoided.
- This spacer arm molecule is insoluble in aqueous reaction conditions and must be dissolved in organic solvents prior to the addition to the aqueous buffered reaction solution.
- the activated chips are then immersed in a 1 mM solution of dithio- bis(succinimidylundecanoate) in 1,4-dioxane for 30-60 minutes at room temperature.
- the chips are then washed three times in 1 ,4-dioxane followed by drying under a stream of nitrogen and immediately used for the immobilization step.
- the activated chips are submerged in a 1 mg/ml solution of the PNA in 0.1 M sodium phosphate, 0.15 M NaCl, at a pH of 7.2 for 30-60 minutes at room temperature, or for several hours at 4°C.
- the chips are then washed three times in the phosphate buffer followed by drying under a stream of nitrogen.
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14203099P | 1999-06-30 | 1999-06-30 | |
US142030P | 1999-06-30 | ||
PCT/US2000/018181 WO2001001144A2 (en) | 1999-06-30 | 2000-06-30 | Hybridization of target dna with immobilized nucleic acid analogs |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1234182A2 true EP1234182A2 (de) | 2002-08-28 |
Family
ID=22498282
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00948560A Withdrawn EP1234182A2 (de) | 1999-06-30 | 2000-06-30 | Hybridisierung von ziel-dna mit immobilisierten nukleinsäureanaloga |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1234182A2 (de) |
AU (1) | AU6203700A (de) |
CA (1) | CA2414329C (de) |
WO (1) | WO2001001144A2 (de) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1263773A4 (de) | 2000-03-14 | 2005-08-31 | Active Motif | Analoge von oligonukleotiden, methoden der synthese und methoden der verwendung |
US7144950B2 (en) | 2003-09-17 | 2006-12-05 | The Regents Of The University Of California | Conformationally flexible cationic conjugated polymers |
US10001475B2 (en) | 2002-06-20 | 2018-06-19 | The Regents Of The University Of California | Light harvesting multichromophore compositions and methods of using the same |
US9371559B2 (en) | 2002-06-20 | 2016-06-21 | The Regents Of The University Of California | Compositions for detection and analysis of polynucleotides using light harvesting multichromophores |
CN1694967B (zh) | 2002-08-26 | 2010-12-15 | 加州大学评议会 | 利用集光多发色团的多核苷酸的检测和分析的方法以及组合物 |
JP4740111B2 (ja) | 2003-02-13 | 2011-08-03 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 集光性多発色団を用いてポリヌクレオチド結合タンパク質相互作用を検出及び分析するための方法並びに組成物 |
WO2005113834A2 (en) | 2004-05-20 | 2005-12-01 | Quest Diagnostics Investments Incorporated | Single label comparative hybridization |
WO2006074482A2 (en) * | 2005-01-10 | 2006-07-13 | The Regents Of The University Of California | Methods and kits for strand-specific polynucleotide detection with cationic multichromophores |
WO2006074471A2 (en) | 2005-01-10 | 2006-07-13 | The Regents Of The University Of California | Cationic conjugated polymers suitable for strand-specific polynucleotide detection in homogeneous and solid state assays |
WO2006081353A2 (en) | 2005-01-27 | 2006-08-03 | Quest Diagnostics Investments Incorporated | Rapid comparative genome hybridization |
WO2006083932A2 (en) | 2005-01-31 | 2006-08-10 | The Regents Of The University | Methods and compositions for aggregant detection |
US8076074B2 (en) | 2005-11-29 | 2011-12-13 | Quest Diagnostics Investments Incorporated | Balanced translocation in comparative hybridization |
US7807359B2 (en) | 2006-12-01 | 2010-10-05 | Quest Diagnostics Investments Incorporated | Methods of detecting TPMT mutations |
US7507539B2 (en) | 2007-07-30 | 2009-03-24 | Quest Diagnostics Investments Incorporated | Substractive single label comparative hybridization |
US8093063B2 (en) | 2007-11-29 | 2012-01-10 | Quest Diagnostics Investments Incorporated | Assay for detecting genetic abnormalities in genomic nucleic acids |
US8039794B2 (en) | 2008-12-16 | 2011-10-18 | Quest Diagnostics Investments Incorporated | Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby |
EP2480684A1 (de) | 2009-09-25 | 2012-08-01 | Signature Genomic Laboratories, Llc | Mehrfach (+/-)-strängige arrays und tests für den nachweis von chromosomalen anomalien im zusammenhang mit krebs und anderen erkrankungen |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU5002996A (en) * | 1995-03-04 | 1996-09-23 | Boehringer Mannheim Gmbh | Sequence-specific detection of nucleic acids |
US5942397A (en) * | 1996-12-11 | 1999-08-24 | Tarlov; Michael J. | Surface immobilization of biopolymers |
US6203758B1 (en) * | 1997-11-10 | 2001-03-20 | Bio-Pixel Ltd. | Micro-circuit system with array of functionalized micro-electrodes |
EP1055004A2 (de) * | 1998-02-20 | 2000-11-29 | Wolfbeis, Otto Samuel | Vorrichtung zur detektion von oligo- und/oder polynukleotid-hybridisierungen |
-
2000
- 2000-06-30 EP EP00948560A patent/EP1234182A2/de not_active Withdrawn
- 2000-06-30 AU AU62037/00A patent/AU6203700A/en not_active Abandoned
- 2000-06-30 WO PCT/US2000/018181 patent/WO2001001144A2/en not_active Application Discontinuation
- 2000-06-30 CA CA2414329A patent/CA2414329C/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO0101144A2 * |
Also Published As
Publication number | Publication date |
---|---|
CA2414329C (en) | 2010-04-13 |
WO2001001144A3 (en) | 2001-05-03 |
WO2001001144A2 (en) | 2001-01-04 |
CA2414329A1 (en) | 2001-01-04 |
AU6203700A (en) | 2001-01-31 |
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