CN1323984A - Gene chip colorating detection method - Google Patents

Abstract

In the method, digoxin or biotin mark is added in the PCR augmentatino course of the sample to be tested and then the PCR product is hybridized with the chip, by adding colour developing liquid to obtain the correspounding signal after hybridizing and combining the antibody with digoxin or biotin mark. The correspounding results will be collected and detected with normal optical scanners scanning.

Description

A kind of color developing detection method of genetic chip

Technical field

The present invention relates to genetic chip, particularly a kind of color developing detection method of genetic chip.

Background technology

DNA chip (genetic chip) is one of great science and technology progress of the tool characteristics of the times that occurred in high-tech area in recent years.To be microelectronics, physics, chemistry, material science intersect the high-tech that combines with life science for it.The DNA chip utilizes the intensification and parallel handling principle of microelectronic chip technology, the biological sample that has the characteristics sequence of biological significance in a large number is solidificated in silicon substrate or on glass in an orderly manner, utilize the DNA chip can obtain or handle a large amount of life-information (comprising the identification, detection in Gene Mutation, gene expression profile detection of gene etc.) apace, it is succeedd in fields such as gene expression, gene mutation, Study on gene polymorphism and gene diagnosises and uses.

The correlation technique of genetic chip comprises: the analytical technology of the technology of preparing of genetic chip, the processing of sample and labelling technique, hybridization and detection technique and genetic chip design and hybridization image etc.

At present, gene chip hybridization result's detection method mainly is fluorescence labeling detection method and isotope labeling detection method, the isotope labeling detection method is because complicated operation, pollution is arranged, obtain data speed and wait shortcoming slowly, progressively substituted by the fluorescence labeling detection method, the fluorescence labeling detection method is little with himself peculiar toxic and side effect, highly sensitive, can various product parallel processing (different fluorescence molecules has different wavelength, available laser confocal scanning instrument scans simultaneously the fluorescence of different wave length and reaches the purpose that various product detect simultaneously) etc. advantage take the course of its own, obtain people from all walks of life's favor.But, thereby restricted the application of chip technology aspect medical clinic applications greatly owing to its signal fetch equipment costs an arm and a leg.China also only has minority scientific research tissue and company to possess the ability of this equipment of outfit at present.

Summary of the invention

The technical problem to be solved in the present invention is the problem that exists in detecting at present genetic chip, a kind of color developing detection technology that does not need expensive detecting instrument is provided, this technology can use cheap ordinary optical scanner to scan on the one hand, and the gained collection of illustrative plates is imported computer carry out graphical analysis and data preparation work, under the less situation of quantity of information, also can use magnifier or bore hole Direct observation on the other hand; Simultaneously in the amplification procedure of sample, digoxin or biotin labeling on primer, are greatly reduced the use cost of chip, thereby can thoroughly improve the present situation that current chip costs an arm and a leg and chip detecting equipment costs an arm and a leg and be difficult to promote.

Technical solution of the present invention is to utilize traditional enzymatic reaction to make hybridization signal be presented in chip surface by substrate colors, promptly in the pcr amplification process of detected sample, add digoxin or biotin labeling, hybridize with chip then, combine with digoxin or biotin labeling with antibody after the hybridization, add colour developing liquid again and just can obtain corresponding signal.

Specifically, a kind of color developing detection method of genetic chip, its characteristics are to comprise the following steps:

1. the polymerase chain reaction (PCR) amplification of testing gene chip, and add digoxin or biotin labeling;

2. the hybridization of digoxigenin labeled PCR product and chip;

3. develop the color and scan detection with general optical scanner.

The processing of sample and the process of mark are:

After the DNA extract 2 μ l of testing sample and 12 μ l PCR mixed liquors are mixed, preparing the DNA sample purpose segment of digoxigenin labeled by PCR thermal cycle process, amplimer is 5 '-AGGACG TGG AGG CGA TCA CA-3 ', 5 '-GGG TTG ACC CGC GCG TA-3 ', primer 5 ' end adds digoxigenin labeled, and amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃, 40 seconds, 58 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 30 times, at last, 72 ℃ were extended 5 minutes; With electrophoresis detection PCR product.

The PCR product of described digoxigenin labeled and the hybridization of chip are:

It is mixed to get amplified production 1 μ l and 9 μ l hybridization solutions, drips in the chip array surface, and covered places hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, and the DNA sample of digoxigenin labeled is combined with specific probe on the chip.

The process of described colour developing and detection is:

1. at room temperature, take out chip, remove cover glass, drop in the washing lotion I shaking table rapidly and swing and wash 10 minutes, chip was put into washing lotion II balance again 1 minute (noting not making chip to become dry);

2. from the washing lotion II, take out chip, absorb unnecessary liquid with thieving paper, other gets 20 μ l antibody-solutions and drips in chip surface, and covered left standstill 30 minutes, antibody is fully combined, the albumen nonspecific binding site on the chip of blockading simultaneously with digoxin or biotin;

3. remove cover glass, absorb unnecessary liquid, put then to swing in the washing lotion II and wash 1 minute, take out chip, absorb unnecessary liquid with thieving paper;

4. get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get 15 μ l colour developing drop then, covered (attention can not have bubble to occur and cover glass can not move), black out standing over night in chip surface;

5. wash out chip surface colour developing liquid with deionized water, airing, visible hybridization signal is the mulberry round dot, with ordinary optical scanning instrument record and analysis result, or uses the magnifier Direct observation.

In the pcr amplification process of described sample DNA to be checked, ground suffering or biotin labeling can be on primer, added, also digoxin or biotin labeling can be on dUTP, added.Advantage of the present invention and effect

(1) the color developing detection method of the present invention detection that can suddenly change has high sensitivity and hybridization specificity.

(2) color developing detection method of the present invention does not need expensive fluorescence detector, only needs general optical scanner.

(3) the sample mark among the present invention is handled, and can adopt to add digoxin or biomarker on the primer, thereby can reduce the use cost of chip greatly.

Description of drawings

Fig. 1 carries out chip detection with development process, with the result of common optical scanner scanning gained.

Fig. 2 carries out chip detection with fluorescent marker method, scans the result of gained with the fluorescence detector of induced with laser.

Embodiment

The invention will be further described below in conjunction with embodiments of the invention and accompanying drawing thereof.

The embodiment of the invention 1 its experiment concrete steps are as follows:

1. DNA chip: self-control;

2. PCR mixed liquor: 200 μ M dATP, 200 μ M dGTP, 200 μ M dCTP, 200 μ M dTTP, 1.25U pfu archaeal dna polymerase, upstream and the downstream primer of each 0.2 μ M, 5 of primer ' end carries out mark with digoxin or biotin.If 5 of primer ' end carries out mark with digoxin or biotin.If primer 5 ' end does not add digoxin or biotin carries out mark, then dTTP concentration is 80 μ M, adds 40 μ M digoxin or biotin labeled dUTP again;

3. EasyHtyb: purchase company in Roche;

4. washing lotion I: 150mM NaCl, 15mM sodium citrate, 0.1% lauryl sodium sulfate (SDS);

5. washing lotion II: 100mM maleic acid (Maleic acid), 150mM NaCl, 0.3% (v/v) Tween22, pH7.5;

6. antibody: 0.15U/ml Anti-Digoxigenin-Ap or Anti-Biotin-Ap (purchasing company), 1% (w/v) Blocking Reagent (purchasing company) in Roche in Roche;

7. equilibrium liquid: 100mM Tris-HCl, l00mM NaCl, pH9.5;

8. liquid develops the color: BCIP/NBT Solution (purchasing the company in Roche).

One, the processing of hepatitis B sample and digoxigenin labeled

1, extracting hepatitis B virus gene group DNA (conventional method: see the SDS one Proteinase K method in " clinical gene magnification experiment guide " book) from the patients serum.

2, above-mentioned DNA extract 2 μ l and 12 μ l PCR mixed liquors are mixed after, prepare the DNA sample purpose segment of digoxigenin labeled by PCR thermal cycle process, amplimer is 5 '-AGGACG TGG AGG CGA TCA CA-3 ', 5 '-GGG TTG ACC CGC GCG TA-3 ', primer 5 ' end adds digoxigenin labeled, and amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃, 40 seconds, 58 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 30 times, at last, 72 ℃ were extended 5 minutes; With electrophoresis detection PCR product.

Two, the hybridization of digoxigenin labeled PCR product and chip

It is mixed to get amplified production 1 μ l and 9 μ l hybridization solutions, drips in the chip array surface, and covered places hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, and the DNA sample of digoxigenin labeled is combined with specific probe on the chip.

Three, chip colour developing and results of hybridization detect

(1) at room temperature, take out chip, remove cover glass, drop in the washing lotion I shaking table rapidly and swing and wash 10 minutes, chip was put into washing lotion II balance 1 minute again.

(2) take out chip from the washing lotion II, absorb unnecessary liquid with thieving paper, other gets 20 μ l antibody-solutions and drips in chip surface, and covered left standstill 30 minutes, and antibody is fully combined with digoxin, the albumen nonspecific binding site on the chip of blockading simultaneously.

(3) remove cover glass, absorb unnecessary liquid, put then to swing in the washing lotion II and wash 1 minute, take out chip, absorb unnecessary liquid with thieving paper.

(4) get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get one 15 μ l colour developing drop then in chip surface, covered, black out standing over night.

(5) wash out chip surface colour developing liquid with deionized water, airing, visible hybridization signal is the mulberry round dot.With ordinary optical scanner (EPSON Perfection 1640SU, 1600dpi) sweep record and analysis result (as shown in Figure 1).

Contrast experiment--fluorescence detection experimental procedure:

One. the processing of sample and fluorescence labeling

In 100 μ l hepatitis B patient serum, extract hepatitis B virus DNA with SDS-Proteinase K method.Get above-mentioned DNA extract of 2 μ l and 12 μ l PCR mixed liquor (200 μ M dATP, 200 μ M dGTP, 200 μ M dCTP, 80 μ M dTTP, 1.25U pfu archaeal dna polymerase, upstream and the downstream primer of each 0.2 μ M, 40 μ M CY5-dUTP) mix after, preparing fluorescently-labeled DNA sample purpose segment by PCR thermal cycle process, amplimer is 5 '-AGG ACG TGG AGG CGA TCACA-3 ', 5 '-GGG TTG ACC CGC GCG TA-3 ', amplification condition is: 94 ℃, 5 minutes, with 94 ℃ 40 seconds, 58 ℃ 40 seconds, 72 ℃ 40 seconds the circulation 30 times, at last, 72 ℃ were extended 5 minutes, with electrophoresis detection PCR product;

Two, the chip hybridization of fluorescence detection and result detect

Good sample solution adds on the chip to get mark, and covered places 45 ℃ of hybridization of wet box 30 minutes, puts 2 * SSC then, washes in the 0.1%SDS solution 5 minutes, and 0.1 * SSC washed in the 0.1%SDS solution 5 minutes, swung in 0.1 * SSC solution and washed several times, dried.

The finish cDNA micro-array chip that dries of hybridization is scanned under proper condition with the laser confocal scanning instrument and detects its hybridization signal, obtain the information (shown in Figure 2) in hepatitis B somatotype and mutational site according to the power of information.

Reach in sum the contrast experiment as can be known, advantage of the present invention and effect are as follows:

(1) the color developing detection method of the present invention detection that can suddenly change has suitable sensitivity and assorted Hand over specificity.

(2) color developing detection method of the present invention does not need expensive fluorescence detector, only needs general optics Scanner can be submitted necessary information.

(3) the sample mark among the present invention is processed, and can adopt to add digoxin or biotin mark on the primer Note, thus the use cost of chip can greatly be reduced.

Claims (5)

1, a kind of color developing detection method of genetic chip is characterized in that comprising the following steps:

1. the polymerase chain reaction (PCR) amplification of testing gene chip, and add digoxin or biotin labeling;

2. the hybridization of digoxigenin labeled PCR product and chip;

3. develop the color and scan detection with general optical scanner.

2, the color developing detection method of genetic chip according to claim 1 is characterized in that the processing of sample and the process of mark are:

After the DNA extract 2 μ l of testing sample and 12 μ l PCR mixed liquors are mixed, preparing the DNA sample purpose segment of digoxigenin labeled by PCR thermal cycle process, amplimer is 5 '-AGGACG TGG AGG CGA TCA CA-3 ', 5 '-GGG TTG ACC CGC GCG TA-3 ', primer 5 ' end adds digoxigenin labeled, and amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃, 40 seconds, 58 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 30 times, at last, 72 ℃ were extended 5 minutes; With electrophoresis detection PCR product.

3, the color developing detection method of genetic chip according to claim 1 is characterized in that the PCR product of described digoxigenin labeled and the hybridization of chip are:

It is mixed to get amplified production 1 μ l and 9 μ l hybridization solutions, drips in the chip array surface, and covered places hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, and the DNA sample of digoxigenin labeled is combined with specific probe on the chip.

4, the color developing detection method of genetic chip according to claim 1 is characterized in that the process of described colour developing and detection is:

1. at room temperature, take out chip, remove cover glass, drop in the washing lotion I shaking table rapidly and swing and wash 10 minutes, chip was put into washing lotion II balance again 1 minute (noting not making chip to become dry);

2. from the washing lotion II, take out chip, absorb unnecessary liquid with thieving paper, other gets 20 μ l antibody-solutions and drips in chip surface, and covered left standstill 30 minutes, antibody is fully combined, the albumen nonspecific binding site on the chip of blockading simultaneously with digoxin or biotin;

3. remove cover glass, absorb unnecessary liquid, put then to swing in the washing lotion II and wash 1 minute, take out chip, absorb unnecessary liquid with thieving paper;

4. get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get 15 μ l colour developing drop then, covered (attention can not have bubble to occur and cover glass can not move), black out standing over night in chip surface;

5. wash out chip surface colour developing liquid with deionized water, airing, visible hybridization signal is the mulberry round dot, with ordinary optical scanning instrument record and analysis result, or uses the magnifier Direct observation.

5, the color developing detection method of genetic chip according to claim 1 is characterized in that in the pcr amplification process of sample DNA to be checked, can add ground suffering or biotin labeling on primer, also can add digoxin or biotin labeling on dUTP.

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