Quick and batch test method for copy number of multi-copy genes of genomes

Abstract

The invention discloses a quick and batch test method for the copy number of multi-copy genes of genomes by virtue of a PCR (polymerase chain reaction) technology. The method comprises the following steps of: respectively carrying out PCR amplification and product purification on some multi-copy genes and some known single-copy gene on a same tissue DNA (deoxyribonucleic acid) sample to test the mass concentration of each gene PCR purified product, converting the mass concentration into molar concentration according to the nucleotide sequence of the product, and preparing the standard curves of the genes by same RT-PCR (reverse transcription-polymerase chain reaction) by taking the PCR product and the tissue DNA sample which are subjected to gradient dilution as a template, thus obtaining the concentration of each gene in the DNA sample. Under the same extraction efficiency, the copy number of the multi-copy genes is the number obtained by dividing the quantity of the multi-copy genes under the same volume by the quantity of the single-copy gene, so that the copy number of seven different multi-copy genes in same biont can be tested by once 96-pore RT-PCR reaction, and the copy number of eleven different multi-copy genes can be tested by once 384-pore RT-PCR reaction. Therefore, the quick and batch test efficiency can be realized.

Description

Technical field: [0001] The invention relates to a gene determination method, in particular to a rapid batch determination method for genome multi-copy gene copy number by using PCR technology and RT-PCR technology. Background technique: [0002] Copy-number variant (CNV), also known as copy-number polymorphism (CNP), is a variation of DNA fragments ranging in size from 1kb to 3Mb, widely distributed in the human genome, covering The total number of nucleotides greatly exceeds the total number of single nucleotide polymorphisms (single nucleotide polymorphisms, SNPs), which greatly enriches the diversity of genetic variation in the genome. The expression and regulation of genes in some specific regions of the genome may have very important biological significance. It has been found that the frequency of CNV is much higher than that of chromosome structure variation, and the total number of nucleotides covered in the entire genome is much greater than that of SNP. Therefore,...

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Problems solved by technology

[0004] The main defects of the prior art are: 1. It takes a long time and consumes a lot of money. The above-mentioned sequencing method and the chip method are expensive and time-consuming, especially the design and customization of the chip require high cost and time; at the same time, the processing of data is cumbersome , the biological hybridization method needs a complete growth cycle to produce offspring, and needs to detect a large number of offspring to obtain statistically significant data; 2. Special equipment and devices are required, such as the above-mentioned Southern blot method that requires radioisotope operations and darkrooms Such infrastructure and conditions also require a large amount of funds to deal with radioactive waste. It is difficult for non-radioactive methods to achieve the sensitivity of detecting a single gene, and the price is expensive; 3. The measurement data is not accurate enough. There are many factors affecting DNA dynamics during operation, such as temperature, electrolyte environment, etc., which have a certain impact on the accuracy of the results; 4. Based on insufficient assumptions, the above real-time PCR method is based on an insufficient assumption, that is, the target gene and the internal reference gene have the same amplification coefficient, but this assumption is generally difficult to establish. Genes with different sequences and lengths have different PCR amplification coefficients. Since PCR amplifies genes in an exponential manner, small differences in PCR amplification coefficients will be in many cases. After a cycle of amplification, it is multiplied, resulting in a huge difference in PCR amplification, which directly affects the calculation of gene copy number. The existing method for quickly determining gene copy number by real-time PCR is based on the TapMan fluorescent probe. This quantitative method The method takes a long time to complete the design and customization of the probe. At the same time, it is difficult to design an ideal TapMan fluorescent probe for some genes. Compared with ordinary fluorescent quantitative PCR, the cost is higher. At the same time, the existing real-time quantitative determination of gene copy The number method needs to mix and dilute the target gene and the single-copy internal reference gene in different proportions, and repeated mixing and dilution of small volumes of liquid will greatly increase the experimental error

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