CN1330155A - Membrane transfer method for testing gene chip - Google Patents
Membrane transfer method for testing gene chip Download PDFInfo
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- CN1330155A CN1330155A CN 01113297 CN01113297A CN1330155A CN 1330155 A CN1330155 A CN 1330155A CN 01113297 CN01113297 CN 01113297 CN 01113297 A CN01113297 A CN 01113297A CN 1330155 A CN1330155 A CN 1330155A
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Abstract
A membrane transfer method for testing gene chip includes such steps as adding digoxin or biotin label in the PCR amplification process of the specimen to be tested, hybridizing the PCR resultant with chip, binding antibody with digoxin or biotin label, and transferring the hybridized result to nylon or cellulose ucetate membrane wetted by colouring developing liquid for higher intensity of hybridized signals and higher test sensitivity.
Description
Technical field
The present invention relates to gene chip, particularly a kind of membrane transfer method for testing of gene chip.
Background technology
Gene chip is the intensification and parallel handling principle that utilizes the microelectronic chip technology, the biological sample that has the characteristics sequence of biological significance in a large number is solidificated in silicon substrate or formation on glass in an orderly manner, utilize gene 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 genetic expression, transgenation, Study on gene polymorphism and gene diagnosises and uses.
When using gene chip, target sample nucleic acid often needs through polymerase chain reaction (PCR, hereinafter to be referred as PCR) amplification or clone or reverse transcription (mRNA), isotropic substance or fluorescent mark then carry out in amplification or reverse transcription process, are broken into the library then, through certain chemical treatment, target sample amplifying nucleic acid and probe carry out selective reaction, be hybridization, then nonspecific DNA is removed in flushing, uses certain test set certification mark signal then.Strong marking signal is then sent in hybridization fully, incomplete hybridization signal a little less than, then detect less than signal if can not hybridize, machine is handled and is obtained required measured signal as calculated.
At present, gene chip hybridization result's detection method mainly contains fluorescent mark detection method and isotopic labeling detection method, the isotopic labeling detection method is because complicated operation, pollution is arranged, obtain data speed and wait shortcoming slowly, progressively substituted by the fluorescent mark detection technique, the fluorescent mark detection method is little with himself peculiar toxic side effect, highly sensitive, can be various product equality handle that (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
For these reasons, a kind of new detection method---development process has appearred in the Application Areas at chip, this method has adopted traditional enzymatic reaction to make hybridization signal be presented in chip surface by substrate colors, its results of hybridization can use cheap ordinary optical scanner to scan on the one hand, and the gained collection of illustrative plates is imported computer carry out image analysis and data preparation work, under the less situation of quantity of information, also can use magnifying glass or bore hole direct viewing on the other hand.Thereby improved 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.But the coloration method in using at present is because a little less than the adsorption of chip surface to substrate, thus hybridization signal a little less than, detection sensitivity is lower than fluorescence detection, when dna content is lower in the sample, then is not easy to be detected.
In light of this situation, the technical theme that will solve of the present invention just provides a kind of membrane transfer method for testing of gene chip, it combines the high-density of biochip and film to chromogenic substrate good adsorption performance, make the strength of signal of hybridization improve greatly, its detection sensitivity and fluorescent method are basic identical, to remedy the deficiency of present biochip development process.
Technical solution of the present invention is:
A kind of diaphragm transfer method for testing of gene chip, its characteristics have been to utilize traditional enzymatic reaction to make hybridization signal be presented in the film surface by substrate colors, thereby can design reading of data with cheap optical scanning, specifically, this method is 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, with the nylon membrane or the nitrocellulose membrane that soak into colour developing liquid results of hybridization is transferred on the film again;
The process of said sample preparation and mark is:
Dna solution 2 μ l that extracting is good and 12 μ l mixed liquid of polymerase chair reaction and 1 TagPlus of unit mix, and prepare the DNA sample purpose segment of digoxigenin labeled at the polymerase chain reaction thermal cycler by following thermal cycling process, and upstream and downstream primer be respectively 5 '-TCA AAAAGT TGC GTG CTG-3 ', amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃, 40 seconds, 55 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 30 times, at last, 72 ℃ were extended 5 minutes.With electrophoresis detection PCR product;
The process of the hybridization of said amplified production and chip is:
It is mixed to get amplified production 1 μ l and 9 μ l hybridization, drips in chip array surface, covered.Place hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, the DNA sample is combined with specific oligonucleotide probe on the chip.
At room temperature, take out chip, remove cover glass, drop among the washing lotion I shaking table rapidly and swing and wash 10 minutes, chip was put into washing lotion II balance 1 minute then, take out chip again, 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 vitamin H.
Remove cover glass, absorb unnecessary liquid, put then to swing among the washing lotion II and wash 1 minute, take out chip, absorb unnecessary liquid with thieving paper.
The process that said transfer printing colour developing and result detect comprises:
Get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get a 1 * 1cm then
2Nylon membrane soak into colour developing liquid bonnet in chip surface, black out left standstill 1 hour.
Take off the nylon membrane of chip surface and high temperature copy dried, with the information on the ordinary optical scanning instrument record nylon membrane.
Said sample preparation and mark are in the polymerase chain reaction (PCR) amplification process of detected sample DNA, add digoxin or biotin labeling on dUTP, or add digoxin or biotin labeling on primer.
Described transfer printing colour developing is to adopt nitrocellulose membrane to soak into colour developing liquid bonnet at chip surface.Static 1 hour of black out, thus hybrid structure is transferred on the film.
Description of drawings
Fig. 1 detects ordinary optical scanner scanning gained result with the chip development process.
Fig. 2 detects ordinary optical scanner scanning gained result with the inventive method.
Fig. 3 detects with fluorescent method, the fluorescence detector scanning gained result of induced with laser.
The specific embodiment
The invention will be further described below in conjunction with embodiments of the invention and accompanying drawing thereof.
The concrete grammar of the membrane transfer method for testing of genetic chip of the present invention is as follows:
1, prepares material and reagent
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 the pfu archaeal dna polymerase, upstream and the downstream primer of each 0.2 μ M, 5 of primer ' end land used height Suffering or biotin carry out mark. If primer does not add digoxin or biotin labeling, then that dTTP is dense Degree changes 80 μ M into, adds in addition 40 μ M digoxin or biotin labeled dUTP;
3. EasyHtyb: be purchased from Roche company;
4. washing lotion I:150mM NaCl, 15mM natrium citricum, 0.1% lauryl sodium sulfate (SDS);
5. washing lotion II:100mM maleic acid (Maleic acid), 150mM NaCl, 0.3% (v/v) Tween 22, pH7.5;
6. antibody: 1U/ml Anti-Digoxigenin-Ap or Anti-Biotin-Ap, 1% (w/v) Blocking Reagent (being purchased from Roche company);
7. equilibrium liquid: 100mM Tris-HCl, 100mM NaCl, pH9.5;
8. nitrite ion: BCIP/NBT Solution (being purchased from Roche company);
9. nylon membrane (being purchased from Roche company).
2, concrete steps
(1) the dna solution 2 μ l that extracting is good and 12 μ l PCR mixed liquors and the 1 Tag Plus of unit (being purchased from Shanghai bio-engineering corporation) mixes in the PCR thermal cycler by following Thermal Cycling Preparation digoxin or biotin labeled DNA sample purpose segment, upstream and downstream primer are respectively 5 '-TGA AAA AGT TGC ATG GTG CTG-3 ', 5 '-CTT TTT CAC CTC TGC CTA ATC-3 ', amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃ 40 seconds, 55 ℃ 40 seconds, 72 ℃ 40 Second, circulation was 30 times, and is last, and 72 ℃ were extended 5 minutes, with electrophoresis detection PCR product;
(2) get respectively amplified production 1 μ l and 9 μ l hybridization solutions and mix, drip in the chip array surface, cover Cover glass places the hybridizing box of 40 ℃ of preheatings to be incubated 20-30 minute, makes DNA sample and chip On the specific oligonucleotide probe combination.
(carrying out under the equal room temperature of following steps)
(3) take out chip, remove cover glass, drop into rapidly among the washing lotion I shaking table and swing and wash 10 minutes, again Chip was put into washing lotion II balance 1 minute. (noting not making chip to become dry)
(4) take out chip from washing lotion II, absorb unnecessary liquid with blotting paper, it is molten that other gets 20 μ l antibody Drop is in chip surface, and covered left standstill 30 minutes, and antibody and digoxin or biotin are filled Divide combination, the albumen nonspecific binding site on the chip of blockading simultaneously.
(5) remove cover glass, absorb unnecessary liquid, put then to swing among the washing lotion II and wash 1 minute, take out Chip is absorbed unnecessary liquid with blotting paper.
(6) get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get then a 1 * 1cm2Nylon membrane infiltrate the nitrite ion bonnet in chip surface, black out left standstill 1 hour.
(7) nylon membrane and the high temperature of taking chip surface off are dried, and with the color development stopping reaction, use normal optical Learn the result on the scanning instrument record film.
Carry out below following experiment:
Test sample treatment and the mark of a chip development process and film transfer printing
Extracting Hepatitis B virus-DNA DNA from the patients serum (conventional method: the Proteinase K cracking, Phenol chloroform recovery method), with dna solution 2 μ l and 12 μ l PCR mixed liquors and 1 of above-mentioned extracting The Tag Plus of unit (being purchased from Shanghai bio-engineering corporation) mix in the PCR thermal cycler by with Lower Thermal Cycling prepares DNA sample purpose segment, upstream and the downstream primer branch of digoxigenin labeled Be not 5 '-TGA AAA AGT TGC ATG GTG CTG-3 ', 5 '-CTT TTT CAC CTC TGC CAT ATC-3 ', amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃ 40 seconds, 55 ℃ 40 seconds, 72 ℃ of circulations in 40 seconds 30 times, last, 72 ℃ were extended 5 minutes, with electrophoresis detection PCR product;
Test hybridization and the washing of two chip development processes and film transfer printing
It is mixed to get amplified production 1 μ l and 9 μ l hybridization solutions, drip in the chip array surface, and covered, Place hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, make special on DNA sample and the chip The combination of property oligonucleotide probe.
At room temperature, take out chip, remove cover glass, drop into rapidly among the washing lotion I shaking table and swing and wash 10 fens Clock was put into chip washing lotion II balance 1 minute again. Take out chip, it is unnecessary to absorb with blotting paper again Liquid, other gets 20 μ l antibody-solutions and drips in chip surface, and covered left standstill 30 minutes, made anti-The abundant combination of body and digoxin or biotin, the albumen non-specific binding position on the chip of blockading simultaneously The point.
Remove cover glass, absorb unnecessary liquid, put then to swing among the washing lotion II and wash 1 minute, take out chip, Absorb unnecessary liquid with blotting paper.
Test the colour developing of three chip development processes and detection
Get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get then 15 μ l Develop the color drop in chip surface, covered, black out left standstill 1 hour.
Wash out the chip surface nitrite ion with deionized water, natural air drying behind the deionized water rinsing is used with general Information (result as shown in Figure 1) on the logical optical scanner sweep record film.
Test the colour developing of four film transfer printing development processes and detection
Get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get then a 1 * 1cm2Nylon membrane infiltrate the nitrite ion bonnet in chip surface, black out left standstill 1 hour.
Take nylon membrane and the high temperature of chip surface off and dry, with the color development stopping reaction, sweep with ordinary optical Retouch the information (result as shown in Figure 2) on the instrument sweep record film.
Test processing and the fluorescence labeling of five samples
Extracting Hepatitis B virus-DNA DNA from the patients serum (conventional method: the Proteinase K cracking, Phenol chloroform recovery method), with dna solution 2 μ l and 12 μ l PCR mixed liquor (the 200 μ M of above-mentioned extracting DATP, 200 μ M dGTP, 200 μ M dCTP, 120 μ M dTTP, the CY3 of 80 μ M) and 1 unit Tag Plus (being purchased from Shanghai bio-engineering corporation) mixes in the PCR thermal cycler by following heat Cyclic process prepares fluorescently-labeled DNA sample purpose segment, and upstream and downstream primer be respectively 5 '-TGA AAA AGT TGC ATG GTG CTG-3 ', 5 '-CTT TTT CAC CTC TGC CAT ATC-3 ', amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃ 40 seconds, 55 ℃ 40 seconds, 72 ℃ 40 Second, circulation was 30 times, and is last, and 72 ℃ were extended 5 minutes, with electrophoresis detection PCR product.
The fluoroscopic examination of experiment six hybridization
It is mixed to get amplified production 1 μ l and 9 μ l hybridization, drip in the chip array surface, and covered, Place hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, make special on DNA sample and the chip The combination of property oligonucleotide probe.
Take out chip, remove cover glass, drop into rapidly among the washing lotion I shaking table and swing and wash 10 minutes. Use General Scanning chip signal analytical system Scanarray 3000 sweep records and analysis result (result as Shown in Figure 3).
Can find out that by Fig. 1, Fig. 2 and Fig. 3 advantage of the present invention and effect are as follows:
(1) the film transfer printing color developing detection method of the present invention detection that can suddenly change has with chip and develops the color The hybridization specificity that method is identical with fluorescence detection. The detection sensitivity of film transfer printing and fluorescence detection Quite, but be higher than the method for chip colour developing far away.
(2) film transfer printing color developing detection method of the present invention does not need expensive fluorescence detector equally, only needs General optical scanner.
(3) film transfer printing color developing detection method of the present invention can be with the higher density chip (greater than 400 points/cm2) Results of hybridization show at film.
Claims (6)
1, a kind of diaphragm transfer method for testing of gene chip, it is characterized in that utilizing traditional enzymatic reaction to make hybridization signal be presented in the film surface by substrate colors, thereby can use cheap optical scanner reading of data, specifically, this method is 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, with the nylon membrane or the nitrocellulose membrane that soak into colour developing liquid results of hybridization is transferred on the film again.
2, the membrane transfer method for testing of gene chip according to claim 1, its spy is characterised in that the process of said sample preparation and mark is:
Dna solution 2 μ l that extracting is good and 12 μ l mixed liquid of polymerase chair reaction and 1 TagPlus of unit mix, the DNA sample purpose segment for preparing digoxigenin labeled at the polymerase chain reaction thermal cycler by following thermal cycling process, upstream and downstream primer be respectively 5 '-TCA AAAATG AGT TGC GTG CTG-3 ', 5 '-CTT TTT CAC CTC TGC CTA ATC-3 ', amplification condition is: at first 94 ℃, 5 minutes, with 94 ℃, 40 seconds, 55 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 30 times, last, 72 ℃ were extended 5 minutes.With electrophoresis detection PCR product.
3, the membrane transfer method for testing of gene chip according to claim 1 is characterized in that the process of the hybridization of said amplified production and chip is:
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 is combined with specific oligonucleotide probe on the chip.
At room temperature, take out chip, remove cover glass, drop among the washing lotion I shaking table rapidly and swing and wash 10 minutes, chip was put into washing lotion II balance 1 minute then, take out chip again, 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 vitamin H;
Remove cover glass, absorb unnecessary liquid, put then to swing among the washing lotion II and wash 1 minute, take out chip, absorb unnecessary liquid with thieving paper.
4, the membrane transfer method for testing of gene chip according to claim 1 is characterized in that the process that said transfer printing colour developing and result detect comprises:
Get 50 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, get a 1 * 1cm then
2Nylon membrane soak into colour developing liquid bonnet in chip surface, black out left standstill 1 hour;
Take the nylon membrane and the high temperature of chip surface off and dry, with the information on the ordinary optical scanning instrument record nylon membrane.
5, the membrane transfer method for testing of gene chip according to claim 1 and 2, it is characterized in that said sample preparation and mark, be in the polymerase chain reaction (PCR) amplification process of detected sample DNA, on dUTP, add digoxin or biotin labeling, or on primer, add digoxin or biotin labeling.
6, according to the membrane transfer method for testing of claim 1 or 4 described gene chips, it is characterized in that described transfer printing colour developing is to adopt nitrocellulose membrane to soak into colour developing liquid bonnet at chip surface, static 1 hour of black out, thus hybrid structure is transferred on the film.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB011132973A CN1156586C (en) | 2001-07-06 | 2001-07-06 | Membrane transfer method for testing gene chip |
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| CNB011132973A CN1156586C (en) | 2001-07-06 | 2001-07-06 | Membrane transfer method for testing gene chip |
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| CN1156586C CN1156586C (en) | 2004-07-07 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812529A (en) * | 2010-04-23 | 2010-08-25 | 中国科学院上海微系统与信息技术研究所 | Nanometer compound probe and detection method thereof for gene chip film transfer print |
| CN101942516B (en) * | 2006-06-28 | 2012-09-26 | 邓兴旺 | Method for detecting specific nucleotide sequence using visual film sensor chip |
| CN103293318A (en) * | 2013-05-22 | 2013-09-11 | 吉林大学 | Method for detecting miRNAs (micro ribonucleic acids) by DIG labeling EDC cross-linking bridging method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297819C (en) * | 2004-09-29 | 2007-01-31 | 南京大渊生物技术工程有限责任公司 | Biological chip quantitative detecting method |
| CN102139853B (en) * | 2011-01-10 | 2013-04-03 | 中国人民解放军第二军医大学 | Biotinylated PDMS (polydimethylsiloxane) membrane microfluidic chip and processing method thereof |
-
2001
- 2001-07-06 CN CNB011132973A patent/CN1156586C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942516B (en) * | 2006-06-28 | 2012-09-26 | 邓兴旺 | Method for detecting specific nucleotide sequence using visual film sensor chip |
| CN101812529A (en) * | 2010-04-23 | 2010-08-25 | 中国科学院上海微系统与信息技术研究所 | Nanometer compound probe and detection method thereof for gene chip film transfer print |
| CN101812529B (en) * | 2010-04-23 | 2013-06-26 | 中国科学院上海微系统与信息技术研究所 | Nanometer compound probe and detection method thereof for gene chip film transfer print |
| CN103293318A (en) * | 2013-05-22 | 2013-09-11 | 吉林大学 | Method for detecting miRNAs (micro ribonucleic acids) by DIG labeling EDC cross-linking bridging method |
| CN103293318B (en) * | 2013-05-22 | 2014-10-29 | 吉林大学 | Method for detecting miRNAs (micro ribonucleic acids) by DIG labeling EDC cross-linking bridging method |
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| CN1156586C (en) | 2004-07-07 |
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