Method for detecting CAH related true and false genes

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a method for detecting CAH related true and false genes. Specifically, the invention discloses the detection methodfor determining the mutation condition of the CAH related genes. The CAH related genes comprise a CYP21A2 gene and a CYP11B1 gene. The method comprises the following steps of S1, extracting DNA in asample and carrying out PCR amplification by using a primer group; S2, detecting an amplification product by using a third-generation sequencing platform; and S3, analyzing the sequencing result to obtain the mutation condition of the CAH related genes. According to the method, the true and false genes are expanded together by designing the primer group, a mutation type can be detected by long-fragment library building sequencing, known CYP21A2 and CYP11B1 gene pathogenic mutations can be detected at the same time, the sensitivity is high, only 1-50ng of DNA is required to be contained in thesample, the operation is convenient, the deletion of long fragments can be detected, the true and false genes can be distinguished, and meanwhile, the unknown chromosome structural variation can be detected.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for detecting true and false genes related to CAH. Background technique [0002] The human genome has about 2.91G bases, and there are some special genes among more than 20,000 genes. These genes have very similar pseudogenes, causing great confusion to existing detection methods. Due to the similarity between true and false genes, erroneous recombination may occur during the synapsis of human cells during meiosis, resulting in the fusion of true and false genes, thus generating new mutations that lead to loss or incomplete expression of true genes. [0003] Congenital adrenal hyperplasia (CAH) is a disease caused by an enzyme defect in the synthesis of adrenal cortex hormones. It is an autosomal recessive genetic disease. The incidence of typical CAH is about 1 / 10000, and the incidence of atypical CAH is about 10 times that of typical, and has ethnic specificity. 21-...

AI Technical Summary

Problems solved by technology

[0009]1. The read length is generally 500-800bp, and the sequencing quality of the poly structure is not good, so it cannot be accurately interpreted;

[0010]2. About 7 pairs of primers need to be designed, the workload is heavy and the operation is cumbersome;

[0011]3. The low-dose genome cannot detect the full-length well, and a large amount of DNA in a sample is required to splicing to the full-length;

[0012]4. The read length of the first-generation sequencing is short, which cannot distinguish between true and false genes, and false genes will affect the interpretation of true gene mutations;

[0013]5. Cannot distinguish between true and false genetic transformation

[0015]1. The amount of data required is large, and low-frequency mutations require a large depth to be detected, and the cost is high;

[0016]2. The read length is short, and the deletion or insertion variation of long fragments cannot be detected;

[0017]3. Since the read length is very short, there is no way to distinguish between true and false genes, so it is impossible to judge whether the mutation in the same region of the true gene and the pseudogene sequence comes from the true gene or the pseudogene is a pseudogene

[0021]1. MLPA is greatly affected by the purity of DNA samples, and has high requirements on the purity of the genome. Impurities in the genome will affect the probe hybridization and ligation reactions in MLPA;

[0022]2. Known MLPA kits only design probes for the current high-frequency deletion mutations for detection, and it is difficult to find unknown mutations;

[0024]4. Unable to distinguish the difference between duplications and deletions caused by true and false gene conversion and true deletion duplications, so that it is impossible to carefully understand its pathogenic mechanism and gene structure;

[0025]5. Intron region cannot be detected;

[0026]6. Inaccurate detection of point mutations

[0029]1. First-generation sequencing verification will introduce pseudogene interference, which cannot distinguish true and false genes well, affecting the accuracy of the results;

[0030]2. Inability to distinguish between true and false gene transformation

[0033]1. This method can only detect a limited number of mutations, and it is difficult to interpret the results if an unknown mutation occurs;

[0034]2. It is difficult to design primers for the full length of the gene to distinguish between true and false genes;

[0035]3. If the first-round PCR product is a pseudogene, the test result will be inaccurate

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