CN1188529C - Nano gold mark silver dyeing detection method of gene chip - Google Patents
Nano gold mark silver dyeing detection method of gene chip Download PDFInfo
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Abstract
The present invention discloses a gold nanoparticle mark silver staining detection method for gene chips, which relates to a detection method for the gene chips, particularly to a detection method for nonradioactive marks and nonfluorescent marks of the gene chips. The detection method comprises the following steps: marking the genes of a sample to be detected with digoxin molecules or biotin molecules to obtain the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) marked with the digoxin molecules or the biotin molecules for hybridization with gene chips after the objective genes of the sample to be detected are extracted; combining gold nanoparticle marked digoxin antibodies with the digoxin molecules, or combining gold nanoparticle marked avidin with the biotin molecules after hybridization; staining the hybridized gene chips with silver staining reagents; using the method of direct microscope observation, CCD (charge-coupled device) signal record, or scan detection with a common optical scanner to obtain corresponding hybridization results; and using relevant software for the further analysis of the results.
Description
Technical field
The nano gold mark silver dyeing detection method of a kind of gene chip of the present invention relates to a kind of nonradioactive labeling, non-fluorescent label detection method of gene chip detection method, particularly a kind of gene chip.
Background technology
DNA chip (gene 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.It is the high-tech of microtronics, physics, chemistry, Materials science and life science cross coupled.Gene chip [claims that again DNA chip, biochip, DNA microprobe array (microarray) are with a large amount of gene probes; be fixed on the solid carrier by specific arrangement mode; it carries out the complementation coupling by the base sequence with detected gene; realizing the molecular recognition of nucleotide sequence, is the important means of obtaining associated biomolecule information efficiently on a large scale.The correlation technique of gene chip comprises: the analytical technology of the technology of preparing of gene chip, the processing of sample and labeling technique, hybridization and detection technique and gene chip design and hybridization image etc.For molecular biology, biomedical research, a mensuration and the analysis that basic prerequisite is a dna sequence dna.Traditional dna sequencing method and sequence analysis method are being carried out in the improved process, are that the biochip technology of representative arises at the historic moment with the gene chip.The prototype of gene chip is that the mid-80 proposes, has multiple different using value, as gene expression profile survey, polymorphism analysis, genomic library mapping, drug screening, medical diagnosis on disease and prediction and sequencing by hybridization etc., it makes that synthetic, fixing highdensity ten hundreds of different types of probe molecules are practical, and by laser co-focusing microscan technology, can carry out in real time hybridization signal, sensitive, detect accurately and analyze.
Biochip technology as specimen preparation, chemical reaction and analyzing and testing etc., is integrated into many discontinuous analytic process related in the life science in the chip by adopting technologies such as microelectronics, micromechanics, makes it serialization, integrated and microminiaturized.The maturation of this technology and application will bring a revolution will in the new millennium life science association areas such as genetic research, medical diagnosis on disease and treatment, new drug discovery and environment protection.
At present, Chang Gui molecular hybridization result's detection method mainly is fluorescent mark detection method and isotopic labeling detection method.The molecular hybridization process of film chip is, detected sample is fixed on the filter membrane, hybridize under certain hybridization solution and temperature with isotropic substance or fluorescently-labeled probe, general all need the long time (4-24h) just can finish the molecular hybridization process, and general each can only detect few in number one to several probes.Gene chip is with the dna probe of known array, solidifies in the surface of substrates such as glass, and detected sample is carried out mark and hybridizes with little integrated array.The isotopic labeling detection method has pollution because complicated operation, obtains data speed and waits shortcoming slowly, progressively substituted by the fluorescent mark detection method, and the gene chip detection not only makes and the testing process parallelization can detect hundreds of gene order simultaneously; And because integrated micro-ization makes probe and detected sample that hybridization is required all greatly reduce, and hybridization time obviously shortens, general molecular hybridization process can be finished in 30min.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.
Summary of the invention
The technical problem to be solved in the present invention is at the expensive problem of present gene chip test set, and providing a kind of does not need expensive detecting instrument and the nano gold mark silver dyeing gene chip detecting technique effective shelf time length of reagent.
Technical solution of the present invention is to utilize up-to-date silver to deposit on nm gold particles to make hybridization signal by being presented in chip surface, promptly in the process of the polymerase chain reaction of detected sample (PCR), add digoxin or biotin labeling, hybridize with chip then, combine with the digoxin or the biotin labeling of nano gold mark with antibody after the hybridization, add silver-colored transfection reagent again and just can obtain corresponding signal.
Specifically, a kind of nano gold mark silver dyeing detection method of a kind of gene chip of gene chip, its characteristics are to comprise the following steps;
1. the purifying of testing gene, can use arbitrarily effectively the nucleic acid purification method as: (1) protease K digesting cracking process, (2) are cracking process, (3) alkaline denaturation, (4) boiling method, (5) sodium iodide method, (6) guanidine isothiocyanate method, (7) guanidinium isothiocyanate-silicon-dioxide method, (8) guanidinium isothiocyanate-glass powder method, (9) Chelex-100 method, (10) urea digestion cracking process, the direct TRAP of (11) whole blood directly, to obtain thymus nucleic acid (DNA) or nucleic acid (RNA); Detailed method is referring to " molecular cloning experiment guide ", scientific publication. author, J. Sa nurse Brooker;
2. purpose thymus nucleic acid to be measured (DNA) or nucleic acid (RNA) carry out gene amplification; Gene amplification method is need select one of following method or their combination for use with border factually: (1) polymerase chain reaction (PCR); (2) post transcription cloning polymerase chain (RT-PCR); (3) amplification of dependence nucleotide sequence (Nucleicacid sequence-based amplification, NASBA), the gene amplification of (4) chip original position;
3. nucleic acid labeling methods is selected one of following method for use: (1) is mixed method and added digoxin or biotin labeled nucleic acid monomer in the polymerase chain reaction of thymus nucleic acid (DNA) or nucleic acid (RNA) or post transcription cloning polymerase chain reaction system, (2) end-labelling carries out the polymerase chain reaction or post transcription cloning polymerase chain (3) the chemical labeling method of thymus nucleic acid (DNA) or nucleic acid (RNA) with digoxin or biotin labeled primer, and this method can directly be carried out mark to the nucleic acid of gene amplification product or purifying;
Thymus nucleic acid (DNA) or nucleic acid (RNA) carry out polymerase chain reaction or post transcription cloning polymerase chain be amplified and mark after be used for and gene chip hybridization; If what select for use is the amplification of chip original position, then this step saves.Use hybridization solution to mix during hybridization, change the ionic strength in the gene amplification product and add encapsulant therein with gene amplification product;
5. according to the nucleic acid markers difference, the hybridization back uses the DigiTAb of nano gold mark fully to combine with digoxin, or uses nano gold mark affinity element (streptavidin or avidin) fully to combine with vitamin H;
6. utilize the silver-colored particle aggregation that forms in the redox reaction of silver-colored transfection reagent on nanometer gold, amplify hybridization signal;
7. use the ordinary optical scanner to scan detection, use microscope to detect, image can be carried out digitizing, and can further analyze results of hybridization image based on CCD.Iff being preliminary observation, then available magnifying glass or ordinary optical microscope direct viewing.
8. the fragment of gene amplification is meant that detected object includes the fragment of (1) conservative gene fragment, (2) transgenation; The perhaps combination of (1) (2).
9. the employed primer of the fragment of gene amplification has conservative gene fragment that can the augmentation detection object at least or comprises the fragment of transgenation.
The treating processes of described sample is: the extracting of testing sample gene, at the sample difference, can select to use protease K digesting cracking process, direct cracking process, alkaline denaturation, boiling method, sodium iodide method, guanidine isothiocyanate method, guanidinium isothiocyanate-silicon-dioxide method, guanidinium isothiocyanate-glass powder method, Chelex-100 method, the direct TRAP of whole blood, urea digestion cracking process; Can be referring to " molecular cloning experiment guide ", Science Press. author, J. Sa nurse Brooker; Can use
The labeling process of described sample, illustrate respectively that according to different methods (1) method of mixing has added digoxin or biotin labeled nucleic acid monomer in the polymerase chain reaction of thymus nucleic acid (DNA) or nucleic acid (RNA) or post transcription cloning polymerase chain reaction system, for example, DIG-16-dUTP, Biotin-11-dUTP; (2) end-labelling when primer is synthetic with digoxin or biotin labeling, the polymerase chain reaction of thymus nucleic acid (DNA) or nucleic acid (RNA), post transcription cloning polymerase chain or rely on the amplification of nucleotide sequence; (3) chemical labeling method, this method can directly be carried out mark to the nucleic acid of gene amplification product or purifying, for example uses BrightStar
TMBut Psoralen-Biotin Nonisotopic Labeling Kit labeled rna, DNA, PCR product and oligonucleotide.
Described crossover process is: get the thymus nucleic acid (DNA) of an amount of mark or nucleic acid (RNA) and add after a certain amount of hybridization solution mixes, drip in the gene chip array surface of having sealed, covered, place with gene chip probes Tm value relevant temperature under hybridizing box insulation 20-30 minute of preheating, digoxin or biotin labeled DNA sample are combined with specific probe on the chip.For thymus nucleic acid (DNA) two strands, pre-sex change can improve signal to noise ratio.
Described nano gold mark process is: at room temperature, take out chip, remove cover glass, with washing in the chip scavenging solution 5 minutes, rock frequently, clean twice, blockade simultaneously albumen nonspecific binding site on the chip, take out chip then, absorb unnecessary liquid with thieving paper, other gets the avidin drips of solution of the DigiTAb of nano gold mark or nano gold mark in chip surface, and covered left standstill about 30 minutes, and the DigiTAb of nano gold mark or the avidin of nano gold mark are fully combined with digoxin or vitamin H.Then with washing in the chip scavenging solution 5 minutes.
The described silver process of dying is: in the gene chip hybridization zone, drip to go up silver-colored transfection reagent, and covered leaves standstill about 30 minutes, wash out chip surface liquid, airing with deionized water.
Described testing process is: hybridization signal is by magnifying glass or visible grey of ordinary optical microscope naked eyes or black round dot, uses based on the microscope of CCD or with ordinary optical scanning instrument record and analytical results.
Advantage of the present invention and effect
1. the nano gold mark silver dyeing detection method of a kind of gene chip of the present invention can carry out gene test, has high sensitivity and hybridization specificity.
2. advantages such as the sample mark among the present invention is handled, and it is with low cost to adopt marking method to have, and the reagent validity period is long, and is easy and simple to handle.
3. can use ordinary optical test set to scan based on CCD (charge coupled device), as use the ordinary optical scanner to scan, or use microscope to scan based on CCD, in some cases, observe with magnifying glass or ordinary optical microscope.
In a kind of nano gold mark silver dyeing detection method of gene chip with digoxin or biotin labeling on primer, have with low costly, and can preserve for a long time, greatly reduce the use cost of chip.
5. using signal display method in a kind of nano gold mark silver dyeing detection method of gene chip is silver-colored particle, and the result is easy to preserve, contrast, and stdn; Advantages such as simultaneously, this method is lower than the envrionment conditions of using the enzyme development process to require, and the reaction times is short.
6. in a kind of nano gold mark silver dyeing detection method of gene chip, use charge-coupled device (CCD) as signal picker, even can use the plain scan instrument to carry out signals collecting.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.
Embodiment
Embodiment 1
Hepatitis B detects the nano gold mark silver dyeing detection method process of a kind of gene chip of gene chip:
1, the self-control hepatitis B detects gene chip;
2, the reagent that provides by test kit carries out the gene extracting with the SDS boiling method; Get and boiled 10 minutes after 20 μ l serum add the lysate mixing, per minute 12000 left the heart 10 minutes;
3, follow according to the used gene segment of chip, carry out gene extracting and gene amplification.Its primer adopts the incidental primer of chip agent box, at 5 ' end mark of one of them primer of every pair of primer vitamin H, as 5 '-biotin-CCTGGTTATCGCTGGATG-3 '; 5 '-AGGGTTCAAATGTATACCC-3 '.Carry out the PCR temperature cycle by the specified condition of chip, at first 95 ℃, sex change 3 minutes; Again with 95 ℃, 30 seconds, 52 ℃, 30 seconds, 72 ℃, circulation in 30 seconds 30 times, last, 72 ℃ were extended 4 minutes; The gene amplification product of vitamin H that obtained mark;
4, getting in the chip agent box confining liquid detects gene chip to hepatitis B and seals;
5, get mark vitamin H gene amplification product 94 ℃ down after the abundant sex change at chilling on ice, it is mixed to get 2ul and 1ul hybridization solution, drip in the chip array surface, covered, place hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, biotin labeled DNA sample is combined with specific probe on the chip.
6, at room temperature, take out chip, remove cover glass, washing is 5 minutes in the scavenging solution that the immersion chip agent box provides, and twice, rock solution frequently, absorb unnecessary liquid with thieving paper; Immersed again in the balance liquid that chip agent box provides 1 minute;
7, getting 5 μ l chip agent boxes provides nano gold mark affinity cellulose solution to drip in chip surface, and covered left standstill 10 minutes, and the affinity element is fully combined with vitamin H; Remove cover glass, absorb unnecessary liquid, place balance liquid washing 1 minute then, take out chip, absorb unnecessary liquid with thieving paper;
8, getting chip agent box provides silver-colored transfection reagent 10 μ l to drip in chip surface covered;
9, provide scavenging solution to clean chip surface with chip agent box, dry up, the round dot of visible hybridization signal gray or black under 10 times of magnifying glasses.Use the ordinary optical scanning instrument record;
10, can use various chips hybridization signal analysis software to the hybridization signal analysis.
Embodiment 2
The nano gold mark silver dyeing testing process of multiple blood-borne diseases detection type gene chip:
1, gets blood-borne diseases detection type gene chip, chip surface has been fixed the detection (as HTLV, HIV, HBV, HCV, HPVS, EV, CMV, EBV, HSV) of several genes and has been used the poly oligonucleotide, oligonucleotide sequence contains testing goal gene conserved sequence, and detected object has RNA pathogenic agent and DNA pathogenic agent;
2, reagent and the using method that provides by test kit carried out the gene extracting; Note, should prevent that in experimentation RNA is by enzymolysis; Use RNase free equipment;
3, get RNA to the RNase free Eppendorf tube that 10uL extracts, add 2uL RT primer, under 70 ℃, act on 10 minutes, make the RNA sex change, put what afterwards on ice;
4, take out a new RT pipe, add 38uL DEPC water, put on ice.In several minutes,, all add in the RNA pipe in the step 3 solution in the RT pipe; 42 ℃ of reactions 45 minutes, 70 ℃ were reacted 10 minutes then, promptly finished anti-record;
5, the primer gene amplification that provides by test kit is provided according to the used gene segment of chip.At 5 of the forward primer of every pair of primer ' end mark vitamin H, as 5 '-biotin-CCTGGTTATCGCTGGATG-3 '; 5 '-AGGGTTCAAATGTATACCC-3 '.Carry out the PCR temperature cycle by the specified condition of chip, at first 95 ℃, sex change 3 minutes; Again with 95 ℃, 30 seconds, 52 ℃, 30 seconds, 72 ℃, circulation in 30 seconds 30 times, last, 72 ℃ were extended 4 minutes; The gene amplification product of vitamin H that obtained mark has then been finished reverse transcription and gene amplification simultaneously for the RNA pathogenic agent;
6, getting in the chip agent box confining liquid seals detecting gene chip;
7, get mark vitamin H gene amplification product 94 ℃ down after the abundant sex change at chilling on ice, it is mixed to get 2ul and 1ul hybridization solution, drip in the chip array surface, covered, place hybridizing box insulation 20-30 minute of 40 ℃ of preheatings, biotin labeled DNA sample is combined with specific probe on the chip.
8, at room temperature, take out chip, remove cover glass, washing is 5 minutes in the scavenging solution that the immersion chip agent box provides, and twice, rock solution frequently, absorb unnecessary liquid with thieving paper; Immersed again in the balance liquid that chip agent box provides 1 minute;
9, getting 5 μ l chip agent boxes provides nano gold mark affinity cellulose solution to drip in chip surface, and covered left standstill 10 minutes, and the affinity element is fully combined with vitamin H; Remove cover glass, absorb unnecessary liquid, place balance liquid washing 1 minute then, take out chip, absorb unnecessary liquid with thieving paper;
10, getting chip agent box provides silver-colored transfection reagent 10 μ l to drip in chip surface covered;
11, provide scavenging solution to clean chip surface with chip agent box, dry up, the round dot of visible hybridization signal gray or black under magnifying glass.Use the ordinary optical scanning instrument record;
12, can use various chips hybridization signal analysis software to the hybridization signal analysis.
Embodiment 3
1, self-control hepatitis B mutation checking gene chip; All mutational sites are arranged in three bases of 3 of forward primer ' end, wherein major part be positioned at 3 ' first base position of end on, 5 ' terminal modified amino, these primers as probe stationary at chip surface;
2, the reagent that provides by test kit carries out the gene extracting with the SDS boiling method; Get and boiled 10 minutes after 20 μ l serum add the lysate mixing, per minute 12000 left the heart 10 minutes;
3, follow according to the used gene segment of chip, carry out gene extracting and gene amplification.Use 2 * HotStar Tag RCRbuffer, 50 μ M dNTPs, 20 μ g/ μ l BSA, 0.1-0.4uM reverse primer and 3u HotStar TagDNA polymerase (Qiagen); Wherein oppositely each primer 5 ' end mark vitamin H, as 5 '-biotin-GGGCATTTGGTGGTCT (G/A) T-3 '; 5 '-biotin-AGGGTTCAAATGTATACCC-3 '.Carry out the PCR temperature cycle by the specified condition of chip, at first 95 ℃, sex change 4 minutes; Again with 95 ℃, 40 seconds, 52 ℃, 40 seconds, 72 ℃, circulation in 40 seconds 35 times, last, 72 ℃ were extended 4 minutes; The gene amplification product of vitamin H that obtained mark, and product is combined in chip surface specifically;
4, at room temperature, take out chip, remove cover glass, washing is 5 minutes in the scavenging solution that the immersion chip agent box provides, and twice, rock solution frequently, absorb unnecessary liquid with thieving paper; Immersed again in the balance liquid that chip agent box provides 1 minute;
5, getting 5 μ l chip agent boxes provides nano gold mark affinity cellulose solution to drip in chip surface, and covered left standstill 10 minutes, and the affinity element is fully combined with vitamin H; Remove cover glass, absorb unnecessary liquid, place balance liquid washing 1 minute then, take out chip, absorb unnecessary liquid with thieving paper;
6, getting chip agent box provides silver-colored transfection reagent 10 μ l to drip in chip surface covered;
7, provide scavenging solution to clean chip surface with chip agent box, dry up, the round dot of visible hybridization signal gray or black under 10 times of magnifying glasses.Use the ordinary optical scanning instrument record;
8, can use various chips hybridization signal analysis software to the hybridization signal analysis.
Claims (8)
1, a kind of nano gold mark silver dyeing detection method of gene chip is characterized in that comprising:
A) use one of following method at purpose thymus nucleic acid to be measured (DNA) in the different specimens or nucleic acid (RNA) method for extracting: (1) protease K digesting cracking process, (2) are cracking process, (3) alkaline denaturation, (4) boiling method, (5) sodium iodide method, (6) guanidine isothiocyanate method, (7) guanidinium isothiocyanate-silicon-dioxide method, (8) guanidinium isothiocyanate-glass powder method, (9) Chelex-100 method, (10) urea digestion cracking process, the direct TRAP of (11) whole blood directly, to obtain thymus nucleic acid (DNA) or nucleic acid (RNA);
B) goal gene mark digoxin or vitamin H, its method can use chemical labeling method or gene amplification method;
C) thymus nucleic acid (DNA) or nucleic acid (RNA) are used for and gene chip hybridization behind digoxin or biotin labeling;
D) the hybridization back uses the DigiTAb of nano gold mark to combine with digoxin, or uses nano gold mark affinity element to combine with vitamin H;
E) use silver-colored transfection reagent that the gene chip after hybridizing is dyeed;
F) use ordinary optical scanner scans detection or uses the microscope based on CCD to detect.
2, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1, it is characterized in that: gene amplification method is meant that the gene segment of detected object makes it quantity by amplification method and increases, amplification method can use one of following method or its to be used in combination, and the one, the polymerase chain reaction; The 2nd, the post transcription cloning polymerase chain; The 3rd, rely on the amplification of nucleotide sequence, the 4th, the gene amplification of chip original position.
3, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1, it is characterized in that: the labeling process of gene amplification method mark digoxin or vitamin H sample is one of following method, the one, in the polymerase chain reaction of thymus nucleic acid (DNA) or nucleic acid (RNA) or post transcription cloning polymerase chain reaction system, added digoxin or biotin labeled nucleic acid monomer; The 2nd, carry out the polymerase chain reaction or the post transcription cloning polymerase chain of thymus nucleic acid (DNA) or nucleic acid (RNA) with digoxin or biotin labeled primer; The 3rd, after gene amplification, directly carry out chemical labeling.
4, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1 is characterized in that: use hybridization solution to mix with gene amplification product during hybridization, change the ionic strength in the gene amplification product and add encapsulant therein.
5, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1 is characterized in that: use chip original position gene amplification method simultaneously to gene increase, mark and hybridization.
6, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1 is characterized in that: the hybridization back uses the DigiTAb of nano gold mark fully to combine with digoxin, or uses nano gold mark affinity element fully to combine with vitamin H.
7, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1 and 2 is characterized in that: the fragment of gene amplification is meant that detected object includes the fragment of (1) conservative gene fragment, (2) transgenation; The perhaps combination of (1) (2).
8, the nano gold mark silver dyeing detection method of a kind of gene chip according to claim 1 and 2 is characterized in that: the employed primer of the fragment of gene amplification has conservative gene fragment that can the augmentation detection object at least or comprises the fragment of transgenation.
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| CNB021131473A CN1188529C (en) | 2002-06-11 | 2002-06-11 | Nano gold mark silver dyeing detection method of gene chip |
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| CNB021131473A CN1188529C (en) | 2002-06-11 | 2002-06-11 | Nano gold mark silver dyeing detection method of gene chip |
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| CN1188529C true CN1188529C (en) | 2005-02-09 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7745180B2 (en) * | 2002-04-24 | 2010-06-29 | Hitachi Chemical Co., Ltd. | Device and method for high-throughput quantification of mRNA from whole blood |
| CN100351392C (en) * | 2002-10-14 | 2007-11-28 | 上海华冠生物芯片有限公司 | Marking probe of nano microparticle and affinity element and its preparation method as well as application |
| CN100335655C (en) * | 2004-09-03 | 2007-09-05 | 中国科学院上海生命科学研究院 | Oligonucleotide micro-array chip for detecting small molecule RNA |
| CN1314814C (en) * | 2004-12-17 | 2007-05-09 | 浙江大学 | Signal detection method for gene chip of oligonucleotide with length more than 50 basic groups |
| RU2305270C2 (en) * | 2005-05-18 | 2007-08-27 | Андрей Алексеевич Климов | Fluorescent nanoscopy method (variants) |
| CN101182579B (en) * | 2007-11-19 | 2010-12-08 | 中国科学院上海微系统与信息技术研究所 | Nanometer detecting probe chip without amplifying genom DNA and detection method |
| CN101250585B (en) * | 2008-03-28 | 2012-01-25 | 广州市搏克生物技术有限公司 | Method for detecting DNA, RNA and ultramicro-amount protein |
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