Method for quickly detecting gene copy number in genome

Abstract

The invention provides a method for quickly detecting a gene copy number in a genome. The method comprises the steps of: 1) selecting a gene with a known copy number in an organism as an internal reference gene, and respectively designing TaqMan PCR (polymerase chain reaction) primers and probes of a target gene and the internal reference gene; 2) respectively amplifying and purifying the target gene and the internal reference gene by PCR, measuring respective weight concentration, and converting the weight concentration into molar concentration according to the length of a nucleic acid fragment; 3) mixing the target gene with the internal reference gene according to different molar concentration ratios to obtain a series of standard products, respectively measuring the threshold value fluorescence cycle numbers of the target gene standard products and the internal reference gene standard products by a real-time PCR apparatus, and making a standard curve based on the difference values of a series of threshold value fluorescence cycle numbers and the logarithms of corresponding molar concentration ratios; and 4) extracting and purifying the DNA (deoxyribose nucleic acid) of the measured organism, respectively measuring the threshold value fluorescence cycle numbers of the target gene and the internal reference gene by the real-time PCR apparatus, calculating the difference values of the threshold value fluorescence cycle numbers, and converting the difference values into the molar concentration ratio (namely the gene copy number ratio) of the target gene to the internal reference gene according to the standard curve.

Description

technical field [0001] The invention belongs to biological DNA detection technology, in particular to a method for rapidly detecting gene copy number in genome. Background technique [0002] In modern scientific research and production practice, we sometimes need to know the gene copy number of an organism accurately. For example, the genetic cause of human spinal muscular atrophy (SMA) is a partial deletion of a pair of SMA genes. We need to know exactly the copy number of its gene in order to make a correct diagnosis. Another example is that with the application of transgenic technology in agricultural production, various countries have adopted strict controls on genetically modified organisms, and researchers must accurately know the copy number of the target gene in the transgenic plant. Currently, there are three methods for determining gene copy number: 1. Biological hybridization method. The transgenic plants are crossed with non-transgenic plants, and the copy num...

AI Technical Summary

Problems solved by technology

[0003] The most obvious defect of the prior art is: 1. It takes a long time and costs a lot

2. Special equipment and devices are required

Non-radioactive methods are difficult and expensive to achieve single-gene sensitivity

3. Based on insufficient assumptions

However, this assumption is generally difficult to establish. The PCR amplification coefficients of genes with different sequences and lengths are different.

Since PCR amplifies genes in an exponential manner, a small difference in PCR amplification coefficient will cause a huge difference in PCR amplification after multiple cycles of amplification, which directly affects the calculation of gene copy number

4. Based on insufficient test conditions

But this is very difficult to achieve for a new species or a new target gene

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