CN101851652A - General multiplex polymerase chain reaction realization method based on microarray chip - Google Patents
General multiplex polymerase chain reaction realization method based on microarray chip Download PDFInfo
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Abstract
The invention relates to a general multiplex polymerase chain reaction realization method based on a microarray chip in the technical field of biomedicine. In the realization method, a microarray chip sample application system is used for respectively applying a plurality of pairs of primers to a plurality of hydrophilic micropores of the microarray chip, then a PCR (Polymerase Chain Reaction) component solution is added into the hydrophilic micropores in a pulling mode or a draining mode; the microarray chip of the microarray chip sample application system is placed on an in-situ PCR instrument or in a centrifugal tube filled with mineral oil for thermal cycle amplification, and finally the microarray chip is placed in the centrifugal tube or is directly used for centrifugally collecting PCR products to be used as a secondary amplification template; secondary amplification is carried out on the secondary amplification template by the plurality of primers for detection so as to realize the general multiplex polymerase chain reaction; and a result of the multiplex polymerase chain reaction is read by adopting agarose gel electrophoresis, polyacrylamide electrophoresis, capillary electrophoresis or a DNA chip.
Description
Technical field
What the present invention relates to is a kind of method of field of biomedicine technology, specifically is a kind of implementation method of the general multiplex polymerase chain reaction based on micro-array chip.
Background technology
Multiplex PCR is a kind of special shape of PCR (Polymerase Chain Reaction, polymerase chain reaction), and its feature is in same reaction system, has two pairs or more primer that different purpose fragments is carried out synchronous amplification.Compared with conventional PCR, multiplex PCR has the multiple target synchronous detection, save time, consumption reagent is few, therefore advantages such as required sample size is few reported (Chamberlain for the first time from 1988, J.S., et al., 1988.Nucleic Acids Res., 16,11141-11156) afterwards, multiplex PCR has been successfully applied to many fields, comprise genetically deficient analysis (Sieber, O.M., et al., 2002.Proc.Natl.Acad.Sci.U.S.A, 99,2954-2958), transgenation and Polymorphism Analysis (Moutou, C., et al., 2002.Eur.J.Hum.Genet., 10,231-238), mRNA quantitative analysis (Zimmermann, K., et al., 1996.Biotechniques, 21,480-484), RNA detects (Jin, L., et al., 1996.Mol.CellProbes, 10,191-200; Zou, S., et al., 1998.J.Clin.Microbiol., 36,1544-1548) and the genome sequence analysis (Tettelin, H., et al., 1999.Genomics, 62,500-507).Aspect the diagnosis of infectious diseases, multiplex PCR is at virus (Druce, J., et al., 2002.J.Clin.Microbiol., 40,1728-1732; Robert, P.Y., et al., 2002.J.Med.Virol., 66,506-511), bacterium (Osek, J., 2002.Lett.Appl.Microbiol., 34,304-310; Sloan, L.M., et al., 2002.J.Clin.Microbiol., 40,96-100), parasite (Harris, E., et al., 1998.J.Clin.Microbiol., 36,1989-1995) and drug-resistance of bacteria (Oliveira, D.C.and Lencastre, H.H.2002.Antimicrob.Agents Chemother., 46, important effect has been brought into play in evaluation 2155-2161), analysis and research aspect.
But in the process of multiplex PCR, the amplification that usually occurs between each target fragment is unbalanced, even false negative occurs, and along with the raising of tuple, this phenomenon is just more outstanding.The reason that causes this phenomenon mainly is many phase mutual interference and competitions to primer, makes that each segmental amplification efficiency differs bigger under the same terms.Difference between the primer can obtain suitable minimizing by design of primers and optimization, thereby makes the segmental peak optimization reaction condition of each purpose, and especially annealing temperature is approaching, but influencing each other between the primer is difficult to eliminate.In addition, other component in the multiplex PCR system all needs suitably to adjust to reach the ideal effect as the case may be.(Henegariu such as Henegariu, O., et al., 1997.BioThchniques, 23,504-511) studied each influence factor of multiplex PCR and proposed detailed multiplex PCR optimization step, through multistep optimization, the multiple PCR of particular combinations can reach comparatively ideal expanding effect, but still be subjected to tuple restriction and change array mode or add new primer to the time generally need to optimize again, this has limited the flexible Application of multiplex PCR.Many researchers has been attempted the multiplex amplification that some other method realizes nucleic acid.(Shum such as Shum, J.and Paul, N., 2009.Anal.Biochem., 388, thereby 266-272) by on primer, introduce special chemically modified group be implemented in progressively discharge in the PCR process work primer suppressed to a certain extent different primers between the formation of primer dimer, have the warm start function simultaneously.But this method does not make the effect of multiplex PCR significantly improve, and makes cost improve greatly because of primer is carried out chemically modified.(Meuzelaar such as Meuzelaar, L.S., et al., 2007.Nat.Methods, 4,837-837) universal sequence of primer by 5 ' end being fixed on the magnetic bead (each magnetic bead only be fixed with a kind of primer to) mixes magnetic bead then and is fixed on the hole wall of microwell plate, proceed step by step pcr amplification then, at first be to obtain the set that two ends all have the target fragment of universal sequence, and then increase by universal primer, in theory, this method can be avoided the phase mutual interference between the primer effectively, any a plurality of target fragment that can increase simultaneously, and be not subjected to the restriction of tuple.But, because being fixed on the magnetic bead, primer is in unbound state, the annealing efficiency of template and Auele Specific Primer descends greatly in the PCR process, thereby needs the annealing (twice spend the night) of long period and be different from the annealing temperature (45 ℃) of conventional PCR.And between amplification, want earlier genomic dna is carried out at random pre-amplification improving the copy number of target fragment, thereby reduce false negative rate.Whole process is more loaded down with trivial details and the cycle is very long, and practicality is relatively poor.To sum up, no matter be that clinical application or scientific research all press for a kind of method of simple general-purpose or instrument to realize multiplex PCR rapidly and efficiently.
Microchip technology with high-throughput, advantage such as integrated in the biological study field in occupation of important position more and more.Chip PCR, promptly be the pcr amplification of reaction platform, because improved surface volume than (surface to volume ratio), so can improve efficiency of heat cycle essential in the PCR process greatly with the microchip, thereby shortened the PCR time greatly, improved amplification efficiency.Simultaneously, chip PCR can realize trace P CR again, so can save sample, improves flux.Though the raising of surface volume ratio makes chip surface enlarge markedly the influence of PCR, can be by suitable finishing (Shaoffner, M.A., et al., 1996.Nucleic Acids Res., 24,375-379) overcome, so chip PCR possesses the potential using value.(Matsubara such as Matsubara, Y., et al., 2004.Anal.Chem., 76,6434-6439) designed a kind of chip that is used for pcr amplification, it on the chip one group of separate microwell array, use little point sample technology and in each micropore, add different samples (template) and identical primer and other PCR reactants, entire chip is positioned under the thermal cycling that PCR needs and reacts then, though this method can realize multiplex PCR and need very little system just can finish, the adding of sample needs special-purpose instrument (micro-array chip point sample system) can not be applied to every field on a large scale equally.(Ramalingam such as Ramalingam, N., et al., 2009.Biomed.Mibrodevices, 11,1007-1020) merged the thought of micro-fluid chip, by forming several reaction chambers on the slide through being combined in of structurized PDMS and slide, it is right to add different primers before reaction chamber forms, template and other PCR reactants then add by micro-fluidic technologies, though this method also can realize multiplex PCR, maximum tuple is lower, and the not reproducible use of chip, whole process operation is very complicated.The present invention is directed to the demand of practical application, can easily realize the pcr amplification of higher tuple and possess efficient, to save advantage subsequent operations simultaneously such as sample very simple, can develop multiple detection kit on its basis, have bigger clinical and scientific research and be worth.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of implementation method of the general multiplex polymerase chain reaction based on micro-array chip is provided.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
The first step, use micro-array chip point sample system are put respectively to several hydrophilic microporous of micro-array chip primer some, then the PCR component solution are joined in the hydrophilic microporous with mode of lifting or drainage way;
Described micro-array chip comprises: substrate, hydrophobic surface and micropore, and wherein: hydrophobic surface is positioned at base top, and several micropores run through with array way and are arranged in the hydrophobic surface.
Described micro-array chip is meant: at first at substrate surface sputter or evaporation one deck gold element layer, remove the gold element zone by photoetching and plasma etch processes successively again, utilize chemical treatment method to make remaining gold element laminar surface present hydrophobic property at last.
Describedly primer is put respectively to several hydrophilic microporous of micro-array chip, specifically be meant: in each hydrophilic microporous, click and enter primer inequality, and in each hydrophilic microporous, add cross-linking reagent some.
Described cross-linking reagent is any one in following three kinds of mixed solutions:
A) weight percent is that the chitosan of 0.1%-1% is dissolved in the acetic acid aqueous solution that pH is 4.5-6.0;
B) weight percent is the aqueous gelatin solution of 0.1%-1%;
C) weight percent is the polyoxyethylene glycol aqueous solution of 0.05%-5%.
The described mode of lifting is meant: micro-array chip is immersed the liquid level that proposes the PCR component solution behind the PCR component solution immediately with even velocity.
Described drainage way is meant: the thin slice that will dip in a small amount of PCR component solution skims over the surface of micro-array chip, and the surface hydrophilicity by micro-array chip loads on the PCR component solution in the hydrophilic microporous.
Second goes on foot, is immersed in the mineral oil at micro-array chip surface coverage one deck mineral oil or with entire chip;
The 3rd step, be positioned over the micro-array chip of micro-array chip point sample system on the original position PCR instrument or put into the centrifuge tube that is filled with mineral oil and carry out the thermal cycling amplification, at last micro-array chip is placed centrifuge tube or direct centrifugal collection PCR product to increase the expansion template as secondary;
The 4th step, will be some primer be increased secondary and expand template and carry out the secondary amplification so that detect, thus the realization general multiplex polymerase chain reaction;
Described primer comprises universal primer and Auele Specific Primer.
The 5th step, adopt agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis or DNA chip to read the multiplex polymerase chain re-action result, specifically be meant: when the difference in twos of the segmental length of each purpose in the multiplex polymerase chain re-action adopts agarose gel electrophoresis to read the multiplex polymerase chain re-action result during greater than 30bp; When comprising the mutational site in smaller or equal to the purpose fragment in 30bp or the multiplex polymerase chain re-action, the difference in twos of the segmental length of each purpose in the multiplex polymerase chain re-action adopt the DNA chip to read the multiplex polymerase chain re-action result.
The present invention combines the advantage of multiplex PCR, trace P CR, the amplification ability that has conventional PCR simultaneously, and parent/hydrophobic character of utilizing microarray surface can be quick and easy the adding detected sample, thereby detect and aspect such as other detection of nucleic acids possesses higher value in clinical disease detection, food safety detection, genetically modified organism.
Description of drawings
Fig. 1 is a workflow diagram of the present invention.
Fig. 2 is the micro-array chip synoptic diagram.
Wherein: 1 substrate, 2 hydrophobic surfaces, 3 micropores.
Fig. 3 is Auele Specific Primer of the present invention and universal primer synoptic diagram;
Wherein: 4 upstream universal primers, 5 upstream Auele Specific Primers, 6 templates, 7 downstream Auele Specific Primers, 8 downstream universal primers.
Fig. 4 is embodiment 1 an effect synoptic diagram.
Wherein: M represents molecular weight standard, and 1 is conventional multiple PCR products, and 2 is the universal PC R product based on microarray.
Fig. 5 is embodiment 2 effect synoptic diagram, the results of hybridization of expression probe chip.
Wherein, array 1-11 is respectively the PCR product of 11 kinds of template correspondences and the result of probe hybridization, and array 12 is a blank.Arranging of the probe of all arrays and fixing contrast is identical, the corresponding purpose Segment A ba of probe difference, Cpn, Eco, FH, Hin, Kpn, Lpn, Pae, Sau, Sma, Spn that each array 1-11 is capable.Every kind of probe has 5 repetitions.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1: duchenne muscular dystrophy (Duchenne muscular dystrophy, DMD) synchronous amplification of 9 exons of gene
Sample is healthy people's a blood, uses day rapid DNA of root biochemical technology company limited to extract the amplification kit of parts and extracts genomic dna.Present embodiment may further comprise the steps:
The first step, use that the micro-array chip point sample is many to be put respectively to several hydrophilic microporous primer, then with the PCR component to lift or drainage way joins in the hydrophilic microporous;
Described micro-array chip point sample is from Boao Biological Co., Ltd, and model is SmartArrayer
TM48.
As shown in Figure 2, described micro-array chip comprises: substrate, hydrophobic surface and micropore, and wherein: hydrophobic surface is positioned at base top, and several micropores run through with array way and are arranged in the hydrophobic surface.
Described micro-array chip prepares in the following manner: with common slide as substrate, at first at its surface sputtering or evaporation one deck gold element layer, remove the gold element zone by photoetching and plasma etch processes successively again, promptly form the hydrophilic microporous part, utilize chemical treatment method to make remaining gold element laminar surface present hydrophobic property at last, promptly finish the structure of default close and distant water structure.
As shown in Figure 3, it is synthetic that all primers are given birth to the worker by Shanghai, passed through the PAGE purifying.
Universal primer: upstream primer 5 ' TGTAAAACGACGGCCAGT 3 ', downstream primer 5 ' CAGGAAACAGCTATGACC 3 '.
Auele Specific Primer: used Auele Specific Primer design (Chamberlain on the basis of existing document in the present embodiment, J.S., et al., 1990, PCR protocols:a guide to methods and applications.Academic Press.New York London, pp 272-281), the specifying information of primer is as follows:
| ??Exon | ??Size(bp) | ??Primer?Sequence |
| ??Exon?45 | ??547 | ??F：TGTAAAACGACGGCCAGTaaacatggaacatccttgtggggac??R：CAGGAAACAGCTATGACCcattcctattagatctgtcgccctac |
| ??Exon?48 | ??506 | ??F：TGTAAAACGACGGCCAGTttgaatacattggttaaatcccaacatg??R：CAGGAAACAGCTATGACCcctgaataaagtcttccttaccacac |
| ??Exon?19 | ??459 | ??F：TGTAAAACGACGGCCAGTgatggcaaaagtgttgagaaaaagtc |
| ??R：CAGGAAACAGCTATGACCttctaccacatcccattttcttcca | ||
| ??Exon?17 | ??416 | ??F：TGTAAAACGACGGCCAGTgactttcgatgttgagattactttccc??R：CAGGAAACAGCTATGACCaagcttgagatgctctcacCTTTTCC |
| ??Exon | ??Size(bp) | ??Primer?Sequence |
| ??Exon?51 | ??388 | ??F：TGTAAAACGACGGCCAGTgaaattggctctttagcttgtgtttc??R：CAGGAAACAGCTATGACCggagagtaaagtgattggtggaaaatc |
| ??Exon?8 | ??360 | ??F：TGTAAAACGACGGCCAGTggcctcattctcatgttctaattag??R：CAGGAAACAGCTATGACCgtcctttacacactttacCTGTTGAG |
| ??Exon?12 | ??331 | ??F：TGTAAAACGACGGCCAGTgatagtgggctttacttacatccttc??R：CAGGAAACAGCTATGACCgaaagcacgcaacataagatacacct |
| ??Exon?44 | ??268 | ??F：TGTAAAACGACGGCCAGTcttgatccatatgcttttacctgca??R：CAGGAAACAGCTATGACCtccatcacccttcagaacctgatct |
| ??Exon?4 | ??196 | ??F：TGTAAAACGACGGCCAGTttgtcggtctctctgctggtcagtg??R：CAGGAAACAGCTATGACCcaaagccctcactcaaacatgaagc |
Described hydrophilic microporous is meant: have the microvoid structure of water-wet behavior on the chip, the part that is had hydrophobic property between each micropore separates.
Described PCR component comprises: dna profiling, archaeal dna polymerase, dNTP (triphosphoric acid dezyribonucleoside), damping fluid etc.
Second goes on foot, is immersed in the mineral oil at micro-array chip surface coverage one deck mineral oil of micro-array chip point sample system or with entire chip, to avoid moisture evaporation;
Described mineral oil is: light mineral oil, and available from U.S. Sigma-Aldrich company.
The 3rd step, be positioned over the micro-array chip of micro-array chip point sample system on the original position PCR instrument or put into the EP pipe that is filled with mineral oil and carry out the thermal cycling amplification, at last chip is placed EP pipe (or directly) centrifugal collection PCR product then.
The concrete operations step is:
A. chip is selected.Use diameter 400 μ m, the about 100 μ m of the degree of depth, the cutting size is 10mmx3.6mm, cleans and hydrophobic treatment through piranha solution.
B. point sample.Use the SmartArrayer of Boao Biological Co., Ltd
TM48 micro-array chip point sample systems.The every pair of primer mixes (concentration is 5 μ M) earlier and is dissolved in the acetic acid,diluted solution of 0.65% chitosan (MW:750K), each some point once, every kind of primer point is made two micropores.
C. add template and other first round PCR reactant.Utilize drainage.Each concentration of component just is added with 0.2%BSA in the reaction system without special optimization.
D. first round pcr amplification.To add excellent chip puts into the EP pipe of the 0.2ml that fills 10 μ l ddH2O and 130 μ l mineral oil at once and carries out pcr amplification.The PCR program is 94 ℃ of 5min; 94 ℃
30s，55℃30s，72℃1min，15cycles；94℃30s，63℃90s，25cycles；63℃5min；
E. centrifugal collection product.The centrifugal 1min of 12000rpm takes out chip then.
The 4th step, with universal primer (identical) above-mentioned PCR product (as dna profiling) is carried out secondary amplification (detecting with convenient) with universal sequence thus realize general multiplex polymerase chain reaction, be specially: adding universal primer and all the other reactive components in original EP pipe.The PCR program is 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 30cycles; 72 ℃ of 5min.
Detected through gel electrophoresis: adopt 1 * TAE as electrophoretic buffer, the concentration of sepharose is 2%, and electrophoresis carries out on the Bio-Rad electrophoresis apparatus, applies 100V voltage, and electrophoresis time is 70 minutes.The molecular weight reference is the DL1000 of TaKaRa.The applied sample amount of molecular weight reference is 5 μ l, and the applied sample amount of PCR product is 10 μ l.
Detected result as shown in Figure 4.The result shows that the multiple PCR method that this invention is told about has versatility, can effectively avoid problem such as unbalanced amplification that primer phase mutual interference in the conventional multiplex PCR and competition cause and whole process not to need special optimization, not limited by tuple.
The multiplex amplification and the chip detection of embodiment 2:11 kind pneumonia pathogenic bacterium cloned plasmids
1. sample:
The cloned plasmids correspondence of 11 kinds of pneumonia pathogenic bacterium (seeing the following form) is as template.Carrier is pGM-T, from sky root biochemical technology company limited.Use plasmid extraction test kit (day root biochemistry) to extract plasmid.
| Numbering | Types of spawn | The title abbreviation | Goal gene |
| ??1 | Acinetobacter bauamnnii | ??Aba | ??adeS |
| ??2 | Streptococcus pneumoniae | ??Spn | ??lytA |
| ??3 | Streptococcus aureus | ??Sau | ??femA |
| ??4 | Pseudomonas aeruginosa | ??Pae | ??oprI |
| ??5 | Germ oligotrophy unit cell | ??Sma | ??StmPr |
| ??6 | Hemophilus influenza | ??Hin | ??ompP6 |
| ??7 | Legionella pneumophilia | ??Lpn | ??mip |
| ??8 | Chlamydia pneumoniae | ??Cpn | ??PstI |
| ??9 | Mycoplasma pneumoniae | ??FH | ??P1 |
| ??10 | Klebsiella bacillus | ??Kpn | ??glnL |
| Numbering | Types of spawn | The title abbreviation | Goal gene |
| ??11 | Intestinal bacteria | ??Eco | ??phoA |
2. primer and probe
It is synthetic that all primers and probe all have Shanghai to give birth to the worker, passed through the PAGE purifying.
Universal primer: upstream primer 5 ' TCACTTGCTTCCGTTGTCC 3 ', downstream primer 5 ' GGTTTCGGAT GTTACAGGCA 3 '.
Auele Specific Primer: the sequence according to each target fragment designs voluntarily by primer-design software Primer Premier 5, and sequence information is as follows:
| ??Name | ??Size(bp) | ??Primer?Sequence |
| ??Aba | ??198 | ??F：TCACTTGCTTCCGTTGTCC?CAATTAACTTCTTAGCCGAAGCAGC??R：GGTTTCGGATGTTACAGGCA?TAGGCGTTCTTAACTCATGTGCGAT |
| ??Eco | ??188 | ??F：TCACTTGCTTCCGTTGTCC?ACTGTCATTACGTTGCGGATTAGGC??R：GGTTTCGGATGTTACAGGCA?GTTATCAGTTGGTGAGTGATGCTGC |
| ??FH | ??151 | ??F：TCACTTGCTTCCGTTGTCC?GGCACGAGTAAAACGGCAAACCGAT??R：GGTTTCGGATGTTACAGGCA?ACCAAACCGGGCAGATCACCTTTAA |
| ??Hin | ??126 | ??F：TCACTTGCTTCCGTTGTCC?TAGCTGGTAAAGGTGTTGATGCTGG??R：GGTTTCGGATGTTACAGGCA?ATTAGTACGCTAACACTGCACGACG |
| ??Kpn | ??144 | ??F：TCACTTGCTTCCGTTGTCC?TGAATATCGCGGGCAAGATAGGAGT??R：GGTTTCGGATGTTACAGGCA?CGCCACTATCGACAGTCAGTTCG |
| ??Lpn | ??197 | ??F：TCACTTGCTTCCGTTGTCC?CAAACCACTTGGCAATACAACAACG??R：GGTTTCGGATGTTACAGGCA?AAGACGCTATGAGTGGCGCTCAATT |
| ??Pae | ??152 | ??F：TCACTTGCTTCCGTTGTCC?ACATTTCCATAACAGCAATCTCCC??R：GGTTTCGGATGTTACAGGCA?TTGAACAAACGACACTCCAACTAC |
| ??Name | ??Size(bp) | ??Primer?Sequence |
| ??Sau | ??127 | ??F：TCACTTGCTTCCGTTGTCCAGTGATAACGAATTTGTAGCACAGG??R：GGTTTCGGATGTTACAGGCA?AATCATGATGGCGAGATTACAGGTA |
| ??Sma | ??152 | ??F：TCACTTGCTTCCGTTGTCCTACACGTGCCGTCCGTACCTGA??R：GGTTTCGGATGTTACAGGCACCATGGAGAGGGTCTTGGGCTC |
| ??Spn | ??233 | ??F：TCACTTGCTTCCGTTGTCCGGTTTGAGGTAGTACCAGCCTGTTC??R：GGTTTCGGATGTTACAGGCATGGAGGAAGCACACAGACGGCAACT |
| ??Cp | ??161 | ??F：TCACTTGCTTCCGTTGTCCGCTTCGGGAACGATTTTGGAAACA??R：GGTTTCGGATGTTACAGGCACTGAAGTTGAGCATATTCGTGAGG |
Be used for the specific probe (every kind template 2) of DNA chip, sequence is as follows:
3. make up the oligonucleotide probe chip
Use the aldehyde radical substrate, available from Boao Biological Co., Ltd.
Probe is dissolved in 50% DMSO solution, and final concentration is 10 μ M.Use SmartArrayer
TM48 micro-array chip point sample instruments (available from Boao Biological Co., Ltd) are provided with the point sample program and carry out point sample, and sample (being probe) is selected on the aldehyde radical substrate, place 60 ℃ of wet boxes then 4 hours.
4. multiplex PCR amplification
According to different test purposes and scheme, be divided into following aspect.
A. the various combination of choosing plasmid increases as template, with the validity and the practicality of checking multiplex PCR.Concrete test procedure is similar to Example 1, and 2 differences are arranged: 1. the used chip of the first step PCR is bigger and chip directly is positioned on the original position PCR instrument reacts (covering one deck mineral oil on the chip to avoid evaporating).Original position PCR instrument is available from eastern KingMax true tumor Science and Technology Ltd..2. the Cy5-dUTP that comprises 20 μ M in the second step reaction system is to carry out fluorescent mark to the PCR product.
B. gradient dilution template is to detect its sensitivity.
5. hybridization and detection
Hybridization: (6X SSC, 10X Denhart solution 0.4%SDS) mix at 1: 1, mixing 10s and centrifugal on eddy mixer for the PCR product of mark and hybridization buffer.Then at 95 ℃ of following sex change 5min, take out and ice bath 3min at least immediately.Get 15 μ L and be added on the off-the-shelf chip, 50 ℃ of hybridization 2-3h.Wash after the end.At first chip is put into 42 ℃ of preheatings washings I (2x SSC, 0.2%SDS) in, vibration washing 3min at cleaning solution II (0.2X SSC) washing 3min, soaks 2～3 times last centrifugal drying more at last in distilled water.
Laser scanning detects: the oligonucleotide probe chip model that hybridization is finished is that the chip scanner of GenePix 4200A scans under proper condition, detects its hybridization signal.
Claims (9)
1. the implementation method based on the general multiplex polymerase chain reaction of micro-array chip is characterized in that, may further comprise the steps:
The first step, use micro-array chip point sample system are put respectively to several hydrophilic microporous of micro-array chip primer some, then the PCR component solution are joined in the hydrophilic microporous with mode of lifting or drainage way;
Second goes on foot, is immersed in the mineral oil at micro-array chip surface coverage one deck mineral oil or with entire chip;
The 3rd step, be positioned over the micro-array chip of micro-array chip point sample system on the original position PCR instrument or put into the centrifuge tube that is filled with mineral oil and carry out the thermal cycling amplification, at last micro-array chip is placed centrifuge tube or direct centrifugal collection PCR product to increase the expansion template as secondary;
The 4th step, will be some primer be increased secondary and expand template and carry out the secondary amplification so that detect, thus the realization general multiplex polymerase chain reaction;
The 5th step, employing agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis or DNA chip are read the multiplex polymerase chain re-action result.
2. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, it is characterized in that, described micro-array chip comprises: substrate, hydrophobic surface and micropore, wherein: hydrophobic surface is positioned at base top, and several micropores run through with array way and are arranged in the hydrophobic surface.
3. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, it is characterized in that, described micro-array chip is meant: at first at substrate surface sputter or evaporation one deck gold element layer, remove the gold element zone by photoetching and plasma etch processes successively again, utilize chemical treatment method to make remaining gold element laminar surface present hydrophobic property at last.
4. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1 is characterized in that, the primer described in the first step and the 4th step comprises universal primer and Auele Specific Primer.
5. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, it is characterized in that, primer is put respectively to several hydrophilic microporous of micro-array chip described in the first step with some, specifically be meant: in each hydrophilic microporous, click and enter primer inequality, and in each hydrophilic microporous, add cross-linking reagent.
6. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 5 is characterized in that described cross-linking reagent is any one in following three kinds of mixed solutions:
A) weight percent is that the chitosan of 0.1%-1% is dissolved in the acetic acid aqueous solution that pH is 4.5-6.0;
B) weight percent is the aqueous gelatin solution of 0.1%-1%;
C) weight percent is the polyoxyethylene glycol aqueous solution of 0.05%-5%.
7. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, its spy is that the mode of lifting described in the first step is meant: micro-array chip is immersed the liquid level that proposes the PCR component solution behind the PCR component solution immediately with even velocity.
8. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, it is characterized in that, drainage way described in the first step is meant: the thin slice that will dip in a small amount of PCR component solution skims over the surface of micro-array chip, and the surface hydrophilicity by micro-array chip loads on the PCR component solution in the hydrophilic microporous.
9. the implementation method of the general multiplex polymerase chain reaction based on micro-array chip according to claim 1, it is characterized in that described the 5th step specifically is meant: when the difference in twos of the segmental length of each purpose in the multiplex polymerase chain re-action adopts agarose gel electrophoresis to read the multiplex polymerase chain re-action result during greater than 30BP; When comprising the mutational site in smaller or equal to the purpose fragment in 30BP or the multiplex polymerase chain re-action, the difference in twos of the segmental length of each purpose in the multiplex polymerase chain re-action adopt the DNA chip to read the multiplex polymerase chain re-action result.
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