End-point method genetic typing detection method for carrying out nano upgrade automatic completion of 1536 reactions

Abstract

The invention relates to the technical field of gene detection, and discloses an end-point method genetic typing detection method for carrying out nano upgrade automatic completion of 1536 reactions. The end-point method genetic typing detection method comprises the following steps of: S1: preparing a sample plate: one SNP locus reaction system of one sample in the sample plate is 2* PARMS master mix 0.5[mu]l, DNA template 0.4 [mu]l and ddH2O 0.1 [mu]l, and according to an SNP locus number which needs to be manufactured by each sample, the Sample-Mix of each sample is configured and is respectively added into a 96-pore plate or a 384-pore plate; and S2: preparing an SNP plate: one SNP locus reaction system of one sample in the SNP plate is 2* PARMS master mix 0.5[mu]l, Primer mix 0.2[mu]l and ddH2O 0.3 [mu]l, and according to a sample number which needs to be manufactured by each SNP locus, the SNP-Mix of each SNP locus is configured and is respectively added into the 96-pore plate or the 384-pore plate. The one-time experiment maximum flux of the end-point method genetic typing detection method is 1536 reactions which can be four or sixteen times of a conventional method, meanwhile, the experiment flux of the end-point method genetic typing detection method is flexible and can meet conventional 96 or 384 reaction fluxes, through tests, an optimal reaction system is 2 [mu]l reaction system, and compared with a conventional qPCR 10 [mu]l reaction system, reagent cost 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. 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 corresponding sa...

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.

Images