A nano-upgrade automatic detection method for genotyping 1536 endpoints

Abstract

The present invention relates to the technical field of gene detection, and discloses a detection method for automatically completing 1536 end-point genotyping at nanoscale, including the following steps: S1, sample plate preparation: one SNP site reaction of one sample in the sample plate System: 2×PARMS master mix 0.5μl, DNA template 0.4μl, ddH 2 O 0.1 μl; configure the Sample-Mix of each sample according to the number of SNP sites required for each sample, and distribute it to a 96-well plate or a 384-well plate; S2, SNP plate preparation: one SNP site for one sample in the SNP plate Reaction system: 2×PARMS master mix 0.5μl, Primer mix 0.2μl; ddH 2 O 0.3 μ l; According to the number of samples that each SNP site needs to do, prepare the SNP-Mix of each SNP site, and distribute it to a 96-well plate or a 384-well plate; the highest throughput of a single experiment of the present invention is up to 1536 reactions, It is 4 times or 16 times that of the conventional method. At the same time, the experimental throughput of this method can be changed flexibly, and it can also meet the conventional 96 or 384 reaction throughput. The best reaction system tested is 2μl reaction system, compared with the conventional qPCR 10μl reaction system, the cost of reagents is saved by 80%.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a detection method for automatic genotyping of 1536 endpoints in nanoscale. Background technique [0002] SNP (single nucleotide polymorphism), single nucleotide polymorphism, is a single nucleotide variation that occurs at certain sites on the genome, and is the most common type of heritable variation on the genome, accounting for all known polymorphisms more than 90 percent. There are a large number of SNPs, and as genetic markers, they are often used in various disease-related gene research, animal and plant breeding, species and individual identification, interpretation of molecular genetic mechanisms of diseases, and screening of targeted therapy sites. [0003] The single nucleotide polymorphism (SNP) detection on the gene locus will choose different genotyping detection methods due to the difference in the number of SNPs and the difference in the number of correspo...

AI Technical Summary

Problems solved by technology

[0004] (1) Conventional qPCR instruments limit the throughput of a single experiment to 96 or 384 reactions. When the sample size or the number of SNPs increases, the experimental throughput becomes the biggest limiting factor

[0005] (2) Conventional qPCR genotyping technology, using the TaqMan probe method, needs to design a pair of specific fluorescent probes and a pair of specific amplification primers for each SNP. When the number of SNP sites to be studied increases, the specificity The high cost of fluorescent probes brings great economic pressure to researchers

[0006] (3) Conventional genotyping methods are only applicable to qPCR systems, and are not compatible with fluorescent detection equipment (such as microplate readers) platforms that cannot be detected in real time

[0007] (4) The amount of DNA used in the sample corresponding to the micro-upgraded reaction system is large. When the sample is difficult to obtain or the DNA yield is low, it is often impossible to obtain the detection results of more sites.

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