CN1182252C - Method for preparing double-stranded nucleic acid on the surface of solid phase support - Google Patents
Method for preparing double-stranded nucleic acid on the surface of solid phase support Download PDFInfo
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- CN1182252C CN1182252C CNB021379459A CN02137945A CN1182252C CN 1182252 C CN1182252 C CN 1182252C CN B021379459 A CNB021379459 A CN B021379459A CN 02137945 A CN02137945 A CN 02137945A CN 1182252 C CN1182252 C CN 1182252C
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Abstract
固相支持物表面双链核酸的制备方法是基础分子生物学及生物医学领域中在固相支持物制备双链核酸的一种独特方法，运用这种方法制备连接双链核酸的固相支持物。该制备方法包括如下步骤：(a)化学合成两种单链寡核苷酸片段，其中一个为通用单链寡核苷酸片段，另外一个为探针单链寡核苷酸片段，(b)在液相反应体系中将该通用单链寡核苷酸片段与探针单链寡核苷酸复性并进行核酸连接酶连接反应，(c)将单分子核酸样品点样到表面被醛基修饰的固相支持物表面，经胺化的脱氧胸腺嘧啶核苷酸将单分子核酸连接在固相支持物表面；(d)寡核苷酸微阵列经核酸聚合酶延伸反应，填平固定的单分子核酸的单链寡核苷酸部分，在支持物表面合成完整的双链核酸分子。
The preparation method of double-stranded nucleic acid on the surface of solid support is a unique method for preparing double-stranded nucleic acid on solid support in the fields of basic molecular biology and biomedicine. This method is used to prepare solid support connected to double-stranded nucleic acid . The preparation method comprises the following steps: (a) chemically synthesizing two kinds of single-stranded oligonucleotide fragments, one of which is a universal single-stranded oligonucleotide fragment, and the other is a probe single-stranded oligonucleotide fragment, (b) In the liquid phase reaction system, the universal single-stranded oligonucleotide fragment and the probe single-stranded oligonucleotide are annealed and subjected to a nucleic acid ligase ligation reaction, (c) spotting the single-molecule nucleic acid sample on the surface covered with aldehyde groups On the surface of the modified solid support, the aminated deoxythymidine nucleotide connects the single molecule nucleic acid on the surface of the solid support; (d) the oligonucleotide microarray undergoes a nucleic acid polymerase extension reaction to fill the immobilized The single-stranded oligonucleotide portion of a single-molecule nucleic acid synthesizes a complete double-stranded nucleic acid molecule on the surface of a support.
Description
One, technical field:
The present invention is a kind of peculiar methods for preparing double-strandednucleic acid in base molecule biology and the biomedical sector at solid support, uses this method preparation to connect the solid support of double-strandednucleic acid.
Two, background technology:
The technology of assembling double-strandednucleic acid has significant application value in fields such as making microarray chip of double-stranded nucleic acid, screening nucleic acid binding protein (as transcription factor) and nucleic acid bound drug on solid support.Particularly the present invention provides a kind of economy, technical system efficiently for the preparation microarray chip of double-stranded nucleic acid.
Biochip mainly is meant the microarray that biologically active substances such as the nucleic acid assembled, protein, cell, small tissue constitute on solid support.Applying biochip can realize treating the having that it's too late and how much carry out accurately of materials such as target nucleic acid molecules, protein molecule, cell, small tissue in the sample product, fast, large information capacity detects.Detected characteristics with microminiaturization, high-throughput and automatization.Biochip has extremely important using value meaning on biological detection, medical science detection and medical diagnosis on disease, drug screening and gene sequencing, therefore be subjected to extensive concern and investment, make it become global novel industry, i.e. a biochip industry.
At present the research and development kind of biochip is tending towards variation gradually, comprise initial exploitation and widespread use gene chip, powerful pressing protein chip, the cell chip with life characteristic and organization chip just come onto stage.The detection that realizes on two aspects genetic expression transcribed and translated to gene chip and protein chip can, in the research of genome and protein group, become extremely important technology platform, simultaneously biomedical sectors such as medical clinic applications, new medicament screen, efficacy analysis have been produced revolutionary impact.
Go up from constituting, gene chip and protein chip are made of single strand dna oligonucleotide molecule and protein molecule respectively, therefore gene chip can only rely on hybridization between the nucleic acid molecule detect target single strand dna in the sample behind nucleic acid amplification have that it's too late what, protein chip mainly the identification by protein target molecule in protein and the sample and combine the detection protein target molecule how much have that it's too late.Therefore gene chip can not detect protein, protein chip also is difficult to detect DNA, this has limited utilization DNA chip directly to proteinic detection, and DNA and proteinic identification and interact is kept and expression of gene is brought into play important role in regulating in genome stability.It mainly is that gel shift is tested (gel shift mobility assay) that tradition is used for the ordinary method of researching DNA and protein interaction in the molecular biology, but this method is big to laboratory sample quantity demand, test length consuming time, analysis efficiency is low, and has the harm of radio-labeling to experimenter and environment.
Microarray chip of double-stranded nucleic acid is a kind of biochip.Microarray chip of double-stranded nucleic acid can overcome the single-chain nucleic acid microarray can not detect proteinic defective, because conjugated protein usually identification of DNA and bonded are double-stranded DNAs, takes this to play a significant role in the expression of gene regulation and control.The preparation of microarray chip of double-stranded nucleic acid is the key of this chip application.Lockhart etc. just proposed to prepare method (the US patent No.5 of double chain DNA probe molecule as far back as 1996 on solid-phase media, 556,752), its basic skills is that the oligonucleotide of two sections base complementrities utilization link molecule is linked together, one section is fixed on the solid-phase media, make the oligonucleotide renaturation of two sections base complementrities by annealing, form the double-strandednucleic acid unit molecule that has prominent ring, be used to detect the DNA binding biomolecules; This method is owing to two sections oligonucleotide of chemosynthesis, so production efficiency is low, and cost is higher.Bulyk etc. proposed the microarray chip of double-stranded nucleic acid notion the earliest in 1999, and utilization nucleic acid polymerase primer extension method prepares double-strandednucleic acid microarray (US.Pat.6,326,489).It to the effect that at first passes through the synthesizing single-stranded oligonucleotide microarray of photo etched mask original position on slide, every single stranded oligonucleotide of synthetic is near 3 of slide ' contain the universal primer anneal sequence, after single stranded oligonucleotide microarray and the universal primer annealing, carry out the polysaccharase primer extension reaction, the synthetic second chain nucleic acid, thus the double-strandednucleic acid microarray formed.The shortcoming of this method is to synthesize the long oligonucleotide that contains primer binding site on the solid-phase media surface, is unfavorable for reducing production costs.
We before invented a kind of double-strandednucleic acid microarray technology of preparing (People's Republic of China's national patent application number: 02112780.8), but its production cost is still higher,
Three, summary of the invention:
(1) goal of the invention
The purpose of this invention is to provide a kind of method of being convenient to solid support surface preparation double-strandednucleic acid under the common equipment condition, and the method that preparation cost is low, the more simple complementary single-chain nucleic acid renaturation of the base mispairing rate of double-strandednucleic acid prepares double-strandednucleic acid is low.Can be used for being worth bigger single base mutation double-strandednucleic acid in the solid support surface production.
(2) technical scheme
The present invention is the preparation method of double-stranded nucleic acid on surface of solid support, it is characterized in that this preparation method comprises the steps: two kinds of single stranded oligonucleotide fragments of (a) chemosynthesis, one of them is general strand oligonucleotide fragment, this single stranded oligonucleotide fragment 5 ' end contains two sections reverse complementary base sequences thereof, 3 ' end contains one section outstanding single stranded oligonucleotide fragment, in the aminating deoxythymidine acid of two sections reverse complementary base sequences thereof midfeather C6; Another one is a probe single stranded oligonucleotide fragment, and this single stranded oligonucleotide fragment 3 ' end contains the one section complete complementary single stranded oligonucleotide of single stranded oligonucleotide fragment base fragment that can go out with general strand oligonucleotide fragment 3 ' distal process; (b) should general strand oligonucleotide fragment and probe single stranded oligonucleotide renaturation in liquid-phase reaction system and carry out the nucleic acid ligase ligation, thus be formed on the monomolecular nucleic acid sample that 3 ' end has hairpin structure; (c) monomolecular nucleic acid sample point sample is arrived the surface by aldehyde group modified solid support surface, monomolecular nucleic acid is connected the solid support surface through aminating deoxythymidine acid; (d) oligonucleotide microarray is filled and led up the single stranded oligonucleotide part of fixed monomolecular nucleic acid through the nucleic acid polymerase extension, at the synthetic complete double chain acid molecule of support surface.Fixed single stranded oligonucleotide fragment comprises Yeast Nucleic Acid and thymus nucleic acid two class nucleic acid; The nucleic acid polymerase that is used to carry out the Nucleotide polyreaction comprises ribonucleic acid polymerase, deoxyribonucleic acid polymerase and ThermoScript II; The Nucleotide that is used to carry out the Nucleotide polyreaction comprises all ribonucleotides, all deoxyribonucleotides and special minor nucleotide; The solid support that is used for fixing single stranded oligonucleotide comprises the material that has rigidity and semi-rigid surface that can be used for surface chemistry research.Being used for the solid support that the present invention prepares double-strandednucleic acid can be solid state chemistry material therefors such as LB film, poly tetrafluoroethylene, PVDF membrane, Polystyrene Film, the glass of chemical films such as polycarbonate, gelinite, surface chemical modification, silicon, gold; Similar groups such as active group such as carboxyl, aldehyde radical, amino, hydroxyl, thiol group are contained on the solid support surface.
(3) technique effect
The double-stranded nucleic acid on surface of solid support preparation method that the present invention proposes is in stdn, have 3 important value in the mass production: the one, the double-stranded nucleic acid on surface of solid support preparation method who proposes relies on the characteristics of the specific general oligonucleotide of synthetic itself and probe single stranded oligonucleotide to carry out annealing of liquid phase complementary sequence and ligation, formation contains the specific unit molecule oligonucleotide of partially double stranded nucleic acid, this specific unit molecule oligonucleotide can rely on the aminating deoxythymidine acid of self-contained C6 to be attached to aldehyde group modified solid support surface, fill and lead up the strand part of fixed unit molecule oligonucleotide again by polymerase elongation reaction, thereby at the synthetic complete double-strandednucleic acid in solid support surface; This method is if be used at solid support surface production double-strandednucleic acid microarray, and the production cost of then greatly telling somebody what one's real intentions are is enhanced productivity; During the double-stranded nucleic acid on surface of solid support that the present invention proposes is produced, only need synthetic multi-usage single-stranded probe oligonucleotide on the disposable synthetic general oligonucleotide basis single-stranded probe oligonucleotide of the conjugated protein binding site of DNA (as contain), just can pass through simple nucleic acid renaturation, nucleic acid connection, chip point sample and four normal experiments of nucleic acid polymerase extension, get final product mass production multi-usage double chain oligonucleotide microarray; The 2nd, the present invention proposes the double-stranded nucleic acid on surface of solid support preparation method and extends synthetic double-strandednucleic acid by nucleic acid polymerase, because the fidelity of nucleic acid polymerase, carrying out property be than the height of chemosynthesis, therefore for long or to contain the nucleic acid fragment of degenerate core thuja acid synthetic significant; The 3rd, the little synthetic method of the double-stranded nucleic acid on surface of solid support that the present invention proposes, can guarantee base mispairing rate extremely low in the synthetic double-strandednucleic acid, this has very important value for the solid support surface single base mutation double-strandednucleic acid that production has more large-scale commerce value; This method can overcome the synthetic cover of high price and but can not have the critical defect of the double-strandednucleic acid of single base mutation feature by the method production of hybridization renaturation at sheet synthetic single-chain nucleic acid complementary single-chain nucleic acid.Protein-bonded research especially has important value to the double-stranded nucleic acid on surface of solid support preparation method that the present invention proposes to DNA.DNA and protein interactions genome stability keep and gene expression regulation in bringing into play important role, SDBP particularly, though this proteinoid only account for the nucleus total protein less than 0.1%, but this proteinoid usually is to comprise that the regulatory gene of transcription factor is with certain space-time, the important trans-acting factor of quantity expression, they are by own special structure such as leucine zipper, sequence-specific double-stranded DNA identification combination in the genomes such as zinc fingers, accurately regulate a class function Expression of Related Genes, therefore to the research in this proteinoid and specific recognition bonded DNA site thereof at molecular biology, genomics reaches in the proteomics that has become the primary study object at present and all occupies important status.Yet the methods such as the gel shift experiment that the research in this proteinoid and specific recognition bonded DNA site thereof is developed in traditional molecular biology at present, DNA enzyme I foot printing test, do not have a kind of high-throughout stdn screening method, and present solid support surface single stranded DNA gene chip and the protein chip that has developed all is difficult to carry out the efficient screening and the evaluation of this proteinoid.The double-stranded nucleic acid on surface of solid support technology of preparing that the present invention proposes is if be used to produce the double-strandednucleic acid microarray, then at a low price efficient production contain the conjugated protein double-strandednucleic acid microarray that is combined into a little of DNA, thereby with the research of double-strandednucleic acid microarray high flux screening SDBP and DNA recognition site thereof in nucleus whole protein class range.The foundation of this research platform has important value for the following problem of research.
The one, the double-stranded nucleic acid on surface of solid support preparation method that utilization the present invention proposes produces the double-strandednucleic acid microarray can go out the conjugated protein a complete set of single base mutation double-stranded DNA of the low density micro-array chip as NF-kB of certain sequence-specific double-stranded DNA by gene chip sample applying instrument stdn efficient production, this chip reacts as NF-kB with the corresponding sequence-specific double-stranded DNA of fluorophor mark is conjugated protein, by of the analysis of obtaining of stdn gene chip scanning instrument to hybridization signal, the base that just can obtain conjugated protein identification of this DNA and bonded sequence-specific double-stranded DNA site constitutes characteristics, finds that those participate in the base and the action rule thereof of DNA and protein identification and bonded most critical.
The 2nd, the double-stranded nucleic acid on surface of solid support preparation method that utilization the present invention proposes produce the double-strandednucleic acid microarray can by the machine point sample prepare contain 6～10 bases the high-density double-stranded DNA micro-array chip of the function sequence that might make up, as contain 8 bases might the combination function sequence the double-stranded DNA microarray of 66536 double-chain probes, contain 10 bases might the combination function sequence the double-stranded DNA microarray of 1048576 double-chain probes, use the full nucleus albumen of such double-stranded DNA micro-array chip and certain cell to react, just can obtain all possible SDBP is listed as its dna binding sequence on the chip in the full nucleus albumen of certain cell identification and combining information, thereby scan the DNA site of the conjugated protein identification of all possible DNA that exists on the chip, scan simultaneously with chip on the DNA site all possible DNA of bonded conjugated protein.This is conjugated protein and the conjugated protein DNA recognition site of DNA is very valuable for high flux screening DNA.
The 3rd, the double-stranded nucleic acid on surface of solid support preparation method that utilization the present invention proposes can prepare suitable density by point sample but contain the two strands of the conjugated protein binding site dna fragmentation of having discovered at present of all DNA
The dna microarray chip, by reacting with full nucleus protein hybridization such as the cell that is in Different Organs tissue, different developmental phases, different pathological status etc., just can obtain with the double-stranded DNA micro-array chip on the conjugated protein expressing information in cells such as Different Organs tissue, different developmental phases, different pathological status of all DNA bonded DNA, thereby disclose their function and mutual relationship; This data message is as if the gene expression information in conjunction with conventional expression type gene chip, just can obtain all possible gene of the conjugated protein participation regulation and control of certain DNA, and different genes can be carried out function and sort out, make the mutual relationship of genetic expression in order, this has very important value for great biological questions such as research biological growth, pathology and molecular medicine diagnosis, treatment, new drug developments.
The double-stranded nucleic acid on surface of solid support preparation method of the uniqueness that the present invention foundes can be used in particular for accurately, synthesize efficiently, economically the double-strandednucleic acid microarray on solid support such as slide, this double-strandednucleic acid microarray not only its preparation basis is compatible fully with domestic and international gene chip technology of preparing, and compatible fully with the reaction of general gene expression chip, test set in application, improved the simplicity of operation of this invention product.Therefore the present invention proposes the new method at solid support surface preparation method double-strandednucleic acid, when being used to produce microarray chip of double-stranded nucleic acid, need be in sheet original position synthetic complex process, only need chemosynthesis one general single stranded oligonucleotide and single-stranded probe oligonucleotide, by annealing, connection and point sample are made into the unit molecule oligonucleotide microarray, again by a polymerase elongation reaction, just the unit molecule oligonucleotide microarray can be transformed into the double-strandednucleic acid microarray, not only be convenient to preparation double-strandednucleic acid microarray under the common facility condition, and preparation cost is lower, the method that the more complementary single-chain nucleic acid renaturation of the base mispairing rate of chip double-strandednucleic acid prepares double-strandednucleic acid is low.The most important thing is that this double-strandednucleic acid microarray preparation method can be used for the bigger single base mutation microarray chip of double-stranded nucleic acid of productive value, and that this is the method that relies on merely complementary single-chain nucleic acid renaturation to prepare double-strandednucleic acid is indeterminable.
Four, description of drawings:
Fig. 1 is a solid support surface synthetic unit molecule double chain oligonucleotide
Fig. 2 is the flow process signal of the synthetic unit molecule double chain oligonucleotide in solid support surface
Five, embodiment
1, the preparation of solid support
Being used for the solid support that the present invention prepares double-strandednucleic acid can be solid state chemistry material therefors such as LB film, poly tetrafluoroethylene, PVDF membrane, Polystyrene Film, the glass of chemical films such as polycarbonate, gelinite, surface chemical modification, silicon, gold; Similar groups such as active group such as carboxyl, aldehyde radical, amino, hydroxyl, thiol group are contained on the solid support surface.
2, the preparation of the chemosynthesis of single stranded oligonucleotide and monomolecular nucleic acid sample
Unit molecule oligonucleotide (accompanying drawing 2D) is annealed (accompanying drawing 2C), is connected to form to design synthesising probing needle single stranded oligonucleotide (accompanying drawing 2A) and general single stranded oligonucleotide (accompanying drawing 2B) with two kinds of oligonucleotide after synthetic.
3, monomolecular nucleic acid sample fixing on the solid support surface
The solid support surface is transferred to by suitable mode such as machine, craft or electrophoresis in the synthetic back of solid state chemistry synthetic unit molecule oligonucleotide, and the unit molecule oligonucleotide is connected to solid support surface (accompanying drawing 2E) under proper condition.
4, monomolecular nucleic acid is at the nucleic acid polymerase reaction on solid support surface
Solid support adds nucleic acid polymerase reaction solution, at optimal temperature reaction appropriate time, forms complete double chain acid molecule.(accompanying drawing 2F).
5, the solid support after the polysaccharase polyreaction is used 2 * SSD, 0.1%SDS and 0.2 * SSD, 0.1%SDS washing successively in 5, simply washs with redistilled water at last, and it is standby to put 4 ℃ of preservations.
Design and synthetic following single stranded oligonucleotide carry out double-strandednucleic acid synthetic feasibility checking of the present invention:
Probe single stranded oligonucleotide: 5 ' HO-AGTTGAGGGGACTTTCCTTTTTTGGCCCGTACGC-OH 3 '
General single stranded oligonucleotide: 5 ' P-GGAATCCCCCTGGGGGATTCCGCGTACG-OH 3 '
(the aminating deoxythymidine acid of T:C6)
Use this single stranded oligonucleotide to carry out following two experiments.
1, fluorescent mark (Cy3) ATP infiltration method confirms the polysaccharase building-up reactions
(1) with top synthetic single stranded oligonucleotide annealing, connection, be connected on the slide that the silanization glutaraldehyde handles by the aminating deoxythymidine of C6 acid point sample and form 3 * 3 arrays, sealing is soaked with sodium borohydride in 4 ℃ of backs of spending the night, and washs slide with redistilled water again.
(2) will boil in the slide boiling water 5 minutes, dry up after being cooled to 55 ℃, rapidly at the nucleic acid hybridization damping fluid of 5 ℃ of preheatings of single stranded oligonucleotide microarray place Dropwise 5, after cover glass covers, the inherent 55 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(3) remove hybridization buffer with the simple washing of redistilled water, slide oligonucleotide microarray place drips the polyreaction damping fluid that contains dGTP, dCTP, dTTP, Cy3-dUTP and archaeal dna polymerase (Klenow enzyme), after cover glass covers, the built-in 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(4) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.(result: fluorescent signal is arranged)
(5) after the slide after will scanning takes out, drip HaeIII and enzyme cutting buffering liquid thereof at slide oligonucleotide microarray place, after cover glass covers, the inherent 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(6) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.(result: no fluorescent signal)
2, fluorescent mark (Cy3) NF-kB combines double-stranded DNA microarray and the conjugated protein identification bonded of the DNA feasibility that confirms the present invention's preparation with the double-stranded DNA microarray
(1) with top synthetic single stranded oligonucleotide annealing, connection, be connected on the slide that the silanization glutaraldehyde handles by the aminating deoxythymidine of C6 acid point sample and form 3 * 3 arrays, sealing is soaked with sodium borohydride in 4 ℃ of backs of spending the night, and washs slide with redistilled water again.
(2) will boil in the slide boiling water 5 minutes, dry up after being cooled to 55 ℃, rapidly at the nucleic acid hybridization damping fluid of 5 ℃ of preheatings of single stranded oligonucleotide microarray place Dropwise 5, after cover glass covers, the inherent 55 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(3) remove hybridization buffer with the simple washing of redistilled water, drip the polyreaction damping fluid that contains dGTP, dCTP, dTTP, dATP and archaeal dna polymerase (Klenow enzyme) at slide oligonucleotide microarray place, after cover glass covers, the built-in 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(4) remove the polyreaction damping fluid with the simple washing of redistilled water, drip NF-kB and the DNA binding buffer liquid that contains the Cy3 mark at slide oligonucleotide microarray place behind the drying slide, after cover glass covers, the built-in 25 ℃ of water bath heat preservations of slide moisture releasing box 30 minutes.
(5) remove the association reaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.(result: fluorescent signal is arranged)
(6) after the slide after will scanning takes out, drip HaeIII and enzyme cutting buffering liquid thereof at slide oligonucleotide microarray place, after cover glass covers, the inherent 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(7) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.(result: no fluorescent signal)
Claims (7)
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