CN1314814C - Signal detection method for gene chip of oligonucleotide with length more than 50 basic groups - Google Patents
Signal detection method for gene chip of oligonucleotide with length more than 50 basic groups Download PDFInfo
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Abstract
The present invention discloses a signal detection method for a gene chip of oligonucleotide of which the length is more than 50 basic groups. The method comprises the following steps: polymerase chain reaction amplification is carried out to a target gene, and the target gene is detected; about 5 mu g of positive products are boiled at the temperature of 100 DEG C for denaturalization and are cooled on ice; after the gene chip used for detection is moistened by a phosphate buffer, a pre-hybridization solution is added for pre-hybridization; subsequently, 5 mu g of prepared denaturalized DNA is added, and hybridization is carried out at the temperature of 60 DEG C; first film washing liquid is used for washing the denaturalized DNA for twice, and second film washing liquid is used for washing the denaturalized DNA; the chip is put into a 1*TE buffer mixed with a 1*SYBR first Green for dyeing, the chip is rinsed for twice by the TE buffer, and the rinsing is repeated for twice; signal detection is carried out under ultraviolet light or a fluorescence microscope. The method of the present invention omits the complicated procedure of labeling a probe, and particularly avoids the harm to a human body and the radioactive contamination to an environment caused by isotope labeling. The method applied can overcome a false positive result probably caused by purely using the polymerase chain reaction amplification, and thus, an obtained result is sensitive, stable and reliable.
Description
Technical field
The present invention is a biological technical field, relates in particular to the signal detecting method of a kind of length greater than the gene chip of the oligonucleotide of 50 bases.
Background technology
From Adams in 1992 foundation of expressed sequence tag (expressed sequence tag) and extensive expressed sequence tagging method is subsequently proposed, and the needs that develop of development of biology and life science, biochip technology is arisen at the historic moment.The essence of gene chip (Biochips) be exactly on the little substrate of area in an orderly manner dot matrix arranged a series of addressable biological identification molecule in certain position ground (R.J.Lipshutz et al. that are fixed in, Bio Techniques 19 (1995), 442-447; S.P.Fodor et al., Nature364 (1993), 555-556; S.P.Fodor et al., Science 251 (1991), 767-773; A.C.Pease et al., Proc.Natl.Acad Sci.USA 91 (1994), 5022-5026).Because initial gene chip mainly is to be used for determined dna sequence, gene expression profile identify (D.J.Lockhart et al., Nature Biotechnol.14 (1996), 1675-1680) and the detection and analysis (the Feriotto G of gene mutation body, et al., Hum Mutat1999; 13:390-400; Hacia J.G.et al., Nat Genet 1999; 21 (suppl l): 42-7; Hacia JG, etal., Nat Genet 1999; 22:164-7; Nilsson P, et al., Biotechniques 1999; 26:308-16), so be called DNA chip or gene chip again.At present, this technology is to extend to non-nucleic acid fields such as immune response, receptors bind.Nowadays gene chip mainly comprises cDNA microarray, oligonucleotide microarray, moving electric microarray and protein chip etc.The utilization biochip technology is carried out the chip utilization hybridization technique and the scanning technique that obtain chip processing, valid data extraction, is analyzed and report gene chip, can obtain corresponding gene expression profile (Gene expression profiling).Gene chip is employed in a lot of fields with relative characteristics such as simple because of having strong property, handiness, susceptibility, as: determined dna sequence, gene pleiomorphism detect, discovery, vaccine and drug research (M.J.Cunningham et al., the J.Pharmacol.and Toxicol.Methods 2000 of new gene; 44,291-300), (PNAS 2000 for P.M.Schenk, et al. for the resistance research of plant; 97:11655-11660).
The development of biochip technology certainly will drive the development of large quantities of relevant art, as the signal detecting method of gene chip.The gene chip signal detecting method mainly comprises with the fluorescent signal detection method of Cy5/Cy3 mark with isotope-labeled signal detecting method at present, and these methods mostly are applied in the signal detection of high-throughout gene chip at present, but no matter use the sort of signal detecting method, all need probe is carried out mark, and use the Cy5/Cy3 fluorescent mark and need carry out twice respectively Cy5 and Cy3, not only can operator's the person be come to harm with isotopic labeling, also can cause nuclear radiation to pollute environment.
Given this, the invention provides the signal detecting method of a kind of length greater than the gene chip of the oligonucleotide of 50 bases.The step of method is: the utilization special primer carries out polymerase chain reaction (PCR) amplification and carries out electrophoresis detection with sepharose goal gene; After 100 ℃ of about 5 μ g of polymerase chain reaction (PCR) amplification positive products boil the 5min sex change, directly put into cooled on ice 3-5min, use in order to probe hybridization; With detect with gene chip with the phosphoric acid buffer (pH7.2) of 0.25M carry out wetting after, the adding prehybridization solution (the 0.25mol/L phosphoric acid buffer, pH7.2,1mmol/L EDTA, pH8.0,7%SDS, 1%BSA), 60 ℃ were carried out prehybridization 1-2 hour; Then add the 5 μ g sex change dna probes (this probe need not to carry out any mark, as isotropic substance, vitamin H etc.) that prepare, hybridized 10-12 hour for 60 ℃; ((pH7.0), 0.1%SDS) washing is 2 times for 0.3mol/L NaCl, 0.03mol/L Trisodium Citrate, and each 20min is with film washing liquid II (0.1 * SSC, 0.1%SDS) washing 15min for 2 * SSC with film washing liquid I; Chip is put into dyeing 5-10min in the 1 * TE damping fluid (10mmo/LTrisCl (pHH8.0), 1mmol/LEDTA (pH8.0)) that is mixed with 1 * SYBR Green I; With TE damping fluid rinsing 5-10min, repeat 2 times; Carrying out signal detection under the 254nm UV-light or under the fluorescent microscope.This method has been save numerous and diverse program of probe being carried out mark, has particularly avoided isotopic labeling to injury that human body caused and the radiocontamination that environment is caused; Use this method and can overcome the false positive results that simple utilization polymerase chain reaction (PCR) amplification may cause, make the gained result sensitiveer, stable, reliable.
Summary of the invention
An object of the present invention is to provide the signal detecting method of a kind of length greater than the gene chip of the oligonucleotide of 50 bases.The step of method is:
1) the utilization special primer carries out polymerase chain reaction (PCR) amplification and carries out electrophoresis detection with sepharose goal gene to be measured, and positive PCR product is standby;
2) after polymerase chain reaction (PCR) amplification product 5 μ g boil 5min for 100 ℃ and carry out sex change, directly put into cooled on ice 3-5min;
3) will detect with gene chip with the phosphoric acid buffer of 0.25M, pH7.2, carry out wetting after, add prehybridization solution, 60 ℃ were carried out prehybridization 1-2 hour; Prehybridization solution is the 0.25mol/L phosphoric acid buffer, pH7.2, and the 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0, weightmeasurement ratio are 7% sodium laurylsulfonate, weightmeasurement ratio is 1% bovine serum albumin;
4) then add step 2) 5 μ g denatured DNAs, hybridized 10-12 hour for 55-65 ℃;
5) with film washing liquid I washing 1-3 time, each 15-20min; Film washing liquid I is 2 * SSC, and weightmeasurement ratio is 0.1% sodium laurylsulfonate; 2 * SSC is a 0.3mol/L sodium-chlor, 0.03mol/L Trisodium Citrate, pH7.0;
6) with film washing liquid II washing 15-20min; Film washing liquid II is 0.1 * SSC, and weightmeasurement ratio is 0.1% sodium laurylsulfonate;
7) gene chip is put into the 1 * TE damping fluid that the is mixed with 1 * SYBR Green I 5-10min that dyes; 1 * TE damping fluid is a 10mmol/L Tris alkali, pH8.0; The 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0;
8) with TE damping fluid rinsing 5-10min, repeat 2-3 time;
9) carrying out signal detection under the 254nm UV-light or under the fluorescent microscope.
Advantage of the present invention is: 1) this method has been save numerous and diverse program of probe being carried out mark, has particularly avoided isotopic labeling to injury that human body caused and the radiocontamination that environment is caused; 2) use this method and can overcome the false positive results that simple utilization polymerase chain reaction (PCR) amplification may cause, make the gained result more reliable.3) this method is compared with the detection method of routine and is had highly sensitive and higher recall rate.
Description of drawings
Fig. 1 is that Nos terminator gene is to carry out painted result behind the probe hybridization, and among the figure: 1 is the Nos terminator, and 2 is the NPTII gene, and 3 is the CP4-EPSPS gene, and 4 is the Bar gene;
Fig. 2 is used for the structural representation of length greater than the signal detecting method gene chip of the gene chip of the oligonucleotide of 50 bases;
Fig. 3 is used for the square formation structure enlarged diagram of length greater than signal detecting method gene chip Fig. 2 of the gene chip of the oligonucleotide of 50 bases.
Embodiment
Embodiment
(1) length is greater than the preparation of the gene chip of the oligonucleotide of 50 bases
As shown in Figure 2, gene chip is the film 6 that is coated with positively charged material on the plane of flush type prop carrier 5, be provided with dot matrix 7 on the surface of this rete, on dot matrix 7, be placed with the following foreign gene of judging usefulness for transgenic plant, 35S promoter, FMV35S promotor and Nos terminator probe, wherein, foreign gene: 1) NPTII gene, 2) CryI (AC) gene, 3) Cry9c gene, 4) the CP4-EPSPS gene of Gai Zaoing, 5) 35s-CTP2,6) CP4-EPSPS gene, 7) GOX gene, 8) Bar gene, 9) Bamase gene, 10) Barstar gene, 11) CryIA (a) gene, 12) Lectin gene, 13) Napin gene, 14) Zein gene, 15) Lat52 gene Sad1 gene and 16).
The recess shape structure 8 of dot matrix 7 of the present invention for arranging, maintain spaced interval between the dot matrix 7, said each foreign gene, 35S promoter, FMV35S promotor and Nos terminator probe for transgenic plant judgement usefulness are coated on respectively in each recess shape structure 8.The rete 6 of positively charged material is polylysine material or aminosilane film layer.Flush type prop carrier 5 is slide, silicon chip, nylon membrane or nitrocellulose filter.It is 0.2 μ g/ μ l that the single stranded oligonucleotide fragment (length is greater than 50bp) of synthesizing good 19 above-mentioned genes in advance is diluted to concentration, according to requirement of experiment with various concentration, various heterogeneic single stranded oligonucleotide arrangement of fragments in 96 hole PCR plates, be used for the some system of gene chip; (GenomicSolution, USA) robot arm use the 96 pin mark systems of diameter as 0.4mm to gene chip, and gene chip carrier is nylon membrane (Millipore Nylon N by arrayer
+), all some points are made as 36 * 24 one-level square formation, and three kinds of different concns of every kind of gene are the parallel repeatability that repeats with proof test for three times in 3 * 3 secondary battle array.
(2) CTAB method extracting transgenic soybean DNA
With liquid nitrogen the 0.1g genetically engineered soybean is clayed into power, the CTAB that pulverous dry-matter is added 400 μ l precoolings extract damping fluid I (50mmol/L Tris-Cl, 10mmol/L EDTA, 800mmol/L NaCl, pH8.0) in.The CTAB that adds 65 ℃ of preheatings of 500 μ l extract damping fluid II (2%CTAB, 50mmol/LTris-Cl, 10mmol/L EDTA, 800mmol/L NaCl, pH8.0), 1 μ l 2 mercapto ethanol, mixing, 65 ℃ of insulation 30-90min, the light and slow frequently therebetween mixing of putting upside down.Add 450 μ l chloroform/primary isoamyl alcohol after waiting to be chilled to room temperature, light and slow mixing to the solution of putting upside down is the milkiness shape.The centrifugal 2min of 12000rpm is to phase-splitting.Supernatant liquor is transferred in the clean centrifuge tube, adds 5 μ l Rnase A liquid storages, be incubated 30min under the room temperature.Add 600 μ l Virahols and 60 μ l sodium acetate solutions successively, the light and slow mixing of putting upside down.12, the centrifugal 10min of 000rpm, deposit D NA.After abandoning supernatant liquor, add 800 μ l, 76% ethanol, 12, the centrifugal 10min of 000rpm, abandon supernatant liquor after, add 100 μ l, 70% washing with alcohol precipitation again, 12, the centrifugal 5min of 000rpm, deposit D NA removes residual ethanol.The DNA resolution of precipitate is in 100 μ l TE damping fluids, and 4 ℃ of preservations are standby.
(3) polymerase chain reaction (PCR) amplification
The total reaction system is 50 μ l, the above-mentioned template DNA of 10 μ l, 5 μ l, 10 * PCR reaction buffer (500mmol/L KCl, 100mmol/L TrisCl, under 25 ℃, pH9.0,1.0%Triton X-100), 4 μ l 25mmol/L MgCl
2, 4 kinds of dNTP of 4 μ l (every kind of 2.5mmol/L), 0.5 μ l upstream primer (100ng/ μ l), 0.5 μ l downstream primer (100ng/ μ l), 1 μ l Taq archaeal dna polymerase (5U/ μ l), redistilled water 25 μ l, behind the mixing centrifugal 5 seconds.With mixture 94 ℃ down ice-cold behind the heating 5min, the rapid centrifugal several seconds, make that drop is sink to the pipe end on the tube wall, add Taq archaeal dna polymerase (the about 2.5U of 0.5 μ l), centrifugal slightly behind the mixing.95 ℃ of sex change 5min, with 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 amplified reaction circulations, carry out PCR.After last took turns loop ends, insulation 10min under 72 ℃ made the reaction product amplification fully.The primer of pcr amplification is respectively:
Nos terminator gene
Pnos-F1：5’GAATCCTGTTGCCGGTCTTG3’
Pnos-R1：5’TTATCCTAGTTTGCGCGCTA3’
(4) agarose gel electrophoresis
(1mmol/L EDTA pH8.0), prepares 1% sepharose liquid for 45mmol/L Tris alkali, 45mmol/L boric acid with 0.5 * TBE electrophoretic buffer; Add 10mg/ml ethidium bromide (EB) solution mother liquor, making its final concentration is 0.5 μ g/ml; Prepare corresponding gel slab with the glue plate; Get 10 μ l PCR products and 2 μ l, 6 * load sample damping fluid (0.25% tetrabromophenol sulfonphthalein, 40% (w/v) aqueous sucrose solution) mixing, carefully add with the micropipette rifle in the sample cell of gel slab; Control voltage remains on 60-80V, and electric current is more than 40mA, and the demand working power supply carries out electrophoresis; When the tetrabromophenol sulfonphthalein band moves to apart from the about 2cm in gel forward position, stop electrophoresis; Be to observe dyeing under the long wavelength ultraviolet lamp of 254nm and take pictures at wavelength.
(5) molecular hybridization and signal detection
Amplified production about 5 μ g in polymerase chain reaction (PCR) boil 5min for 100 ℃ carry out sex change after, directly put into cooled on ice 3-5min, use in order to probe hybridization; With detect with gene chip with the phosphoric acid buffer (pH7.2) of 0.25M carry out wetting after, the adding prehybridization solution (the 0.25mol/L phosphoric acid buffer, pH7.2,1mmol/L EDTA, pH8.0,7%SDS, 1%BSA), 60 ℃ were carried out prehybridization 1-2 hour; Then add the 5 μ g sex change dna probes (this probe does not carry out any mark, as isotropic substance, vitamin H etc.) that prepare, hybridized 10-12 hour for 60 ℃; ((pH7.0), 0.1%SDS) washing is 2 times, each 20min for 0.3mol/L NaCl, 0.03mol/L Trisodium Citrate for 2 * SSC with film washing liquid I; With film washing liquid II (0.1 * SSC, 0.1%SDS) washing 15min; Chip is put into dyeing 5-10min in the 1 * TE damping fluid (10mmo/L TrisCl (pH8.0), 1mmol/L EDTA (pH8.0)) that is mixed with 1 * SYBR Green I; With TE damping fluid rinsing 5-10min, repeat 2 times; Carrying out signal detection under the 254nm UV-light or under the fluorescent microscope.
Claims (7)
1. a length is characterized in that greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases the step of method is:
1) the utilization special primer carries out polymerase chain reaction (PCR) amplification and carries out electrophoresis detection with sepharose goal gene to be measured, and positive PCR product is standby;
2) after polymerase chain reaction (PCR) amplification product 5 μ g boil 5min for 100 ℃ and carry out sex change, directly put into cooled on ice 3-5min;
3) will detect with gene chip with the phosphoric acid buffer of 0.25M, pH7.2, carry out wetting after, add prehybridization solution, 60 ℃ were carried out prehybridization 1-2 hour; Prehybridization solution is the 0.25mol/L phosphoric acid buffer, pH7.2, and the 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0, weightmeasurement ratio are 7% sodium laurylsulfonate, weightmeasurement ratio is 1% bovine serum albumin;
4) then add step 2) 5 μ g denatured DNAs, hybridized 10-12 hour for 55-65 ℃;
5) with film washing liquid I washing 1-3 time, each 15-20min; Film washing liquid I is 2 * SSC, and weightmeasurement ratio is 0.1% sodium laurylsulfonate; 2 * SSC is a 0.3mol/L sodium-chlor, 0.03mol/L Trisodium Citrate, pH7.0;
6) with film washing liquid II washing 15-20min; Film washing liquid II is 0.1 * SSC, and weightmeasurement ratio is 0.1% sodium laurylsulfonate;
7) gene chip is put into the 1 * TE damping fluid that the is mixed with 1 * SYBR Green I 5-10min that dyes; 1 * TE damping fluid is a 10mmo/L Tris alkali, pH8.0; The 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0;
8) with TE damping fluid rinsing 5-10min, repeat 2-3 time;
9) carrying out signal detection under the 254nm UV-light or under the fluorescent microscope.
2. a kind of length according to claim 1 is greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases, it is characterized in that, said polymerase chain reaction is: the total reaction system is 50 μ l, 10 μ l template DNAs, 5 μ l, 10 * polymerase chain reaction damping fluid, 4 μ l 25mmol/L MgCl
2, 4 kinds of dNTP of 4 μ l, 0.5 μ l 100ng/ μ l upstream primer, 0.5 μ l 100ng/ μ l downstream primer, 1 μ l 5U/ μ l Taq archaeal dna polymerase, redistilled water 25 μ l, behind the mixing centrifugal 5 seconds; L0 * polymerase chain reaction damping fluid is a 500mmol/L Repone K, 100mmol/L Tris alkali, and pH9.0, weightmeasurement ratio are 1.0%Triton X-100; 4 kinds of dNTP respectively are 2.5mmol/L dATP, dCTP, dGTP, dTTP for comprising; With mixture 94 ℃ down ice-cold behind the heating 5min, the rapid centrifugal several seconds, make that drop is sink to the pipe end on the tube wall, add 2.5U Taq archaeal dna polymerase, centrifugal slightly behind the mixing; 95 ℃ of sex change 5min, with 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 amplified reaction circulations, carry out PCR, after last takes turns loop ends, are incubated 10min down in 72 ℃.
3. a kind of length according to claim 1 is greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases.It is characterized in that said agarose gel electrophoresis method for detecting is: be 1% sepharose liquid with 0.5 * TBE electrophoretic buffer preparation weightmeasurement ratio; 0.5 * TBE electrophoretic buffer is a 45mmol/L Tris alkali, 45mmol/L boric acid, 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0; Add 10mg/ml ethidium bromide solution mother liquor, making its final concentration is 0.5 μ g/ml; Prepare corresponding gel slab with the glue plate; Get 10 μ l PCR products and 2 μ l, 6 * load sample damping fluid mixing; 6 * load sample damping fluid is that weightmeasurement ratio is 0.25% tetrabromophenol sulfonphthalein, and weightmeasurement ratio is 40% aqueous sucrose solution; Add with the micropipette rifle in the sample cell of gel slab; Control voltage remains on 60-80V, and electric current is connected power supply and carried out electrophoresis at 40-100mA; When the tetrabromophenol sulfonphthalein band moves to apart from gel forward position 2-3cm, stop electrophoresis; Be to observe dyeing under the long wavelength ultraviolet lamp of 254nm and take pictures at wavelength.
4. a kind of length according to claim 1 is greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases, it is characterized in that, described gene chip is the film (6) that is coated with positively charged material on the plane of flush type prop carrier (5), be provided with dot matrix (7) on the surface of this rete, on dot matrix (7), be placed with the following foreign gene of judging usefulness for transgenic plant, 35S promoter, FMV35S promotor and Nos terminator probe, wherein, foreign gene is: 1) NPTII gene, 2) CryI (AC) gene, 3) Cry9c gene, 4) the CP4-EPSPS gene of Gai Zaoing, 5) 35s-CTP2,6) CP4-EPSPS gene, 7) GOX gene, 8) Bar gene, 9) Barnase gene, 10) Barstar gene, 11) CryIA (a) gene, 12) Lectin gene, 13) Napin gene, 14) Zein gene, 15) Lat52 gene Sad1 gene and 16).
5. a kind of length according to claim 4 is greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases, it is characterized in that the recess shape structure (8) of described dot matrix (7) for arranging, dot matrix maintains spaced interval between (7), and said each foreign gene, 35S promoter, FMV35S promotor and Nos terminator probe for transgenic plant judgement usefulness are coated on respectively in each recess shape structure (8).
6. a kind of length according to claim 4 is characterized in that greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases the rete of described positively charged material (6) is polylysine material or aminosilane film layer.
7. a kind of length according to claim 4 is characterized in that greater than the signal detecting method of the gene chip of the oligonucleotide of 50 bases described flush type prop carrier (5) is slide, silicon chip, nylon membrane or nitrocellulose filter.
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| CN101595231A (en) | 2006-12-29 | 2009-12-02 | 霍尼韦尔国际公司 | The reaction buffer that is used for microarray |
| CN101074950B (en) * | 2007-06-26 | 2011-02-09 | 东南大学 | Method and apparatus for inspecting gel chip |
| CN102759628B (en) * | 2012-07-05 | 2014-09-24 | 浙江大学 | Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip |
| CN103898229B (en) * | 2014-04-16 | 2016-03-23 | 杭州希诺生物科技有限公司 | SYBR Green I fluorescence dye is used to carry out the Lateral Flow Strip of specific DNA detection |
| CN105733842B (en) * | 2016-03-21 | 2018-10-19 | 珠海国际旅行卫生保健中心 | A kind of gene chip probes stripper |
| CN106198479A (en) * | 2016-08-26 | 2016-12-07 | 周辉 | The SYBR Green I immue quantitative detection reagent box of a kind of detection by quantitative DNA content and detection method thereof |
| CN109745934B (en) * | 2019-03-18 | 2023-11-21 | 中国人民解放军军事科学院军事医学研究院 | Array type synthesizer and inkjet synthesizer |
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| CN1384210A (en) * | 2002-05-14 | 2002-12-11 | 中国科学院武汉病毒研究所 | Detection method of enzyme labelling signal in gene chip |
| CN1392269A (en) * | 2002-06-11 | 2003-01-22 | 刘全俊 | Nano gold mark silver dyeing detection method of gene chip |
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| CN1384210A (en) * | 2002-05-14 | 2002-12-11 | 中国科学院武汉病毒研究所 | Detection method of enzyme labelling signal in gene chip |
| CN1392269A (en) * | 2002-06-11 | 2003-01-22 | 刘全俊 | Nano gold mark silver dyeing detection method of gene chip |
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