PCR (polymerase chain reaction) detection kit for CTG (cytotoxic T G) region of atrophic myotonin kinase gene and application of PCR detection kit

Abstract

The invention provides a PCR (polymerase chain reaction) detection kit for a CTG (cytotoxic T-G) region of an atrophic myotonic protein kinase gene. The PCR detection kit comprises specific primer pairs with nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. The method has the characteristics of strong amplification specificity and small amplification preference. And meanwhile, the technical difficulty that the false negative is displayed when part of samples are detected by the existing method is overcome, and the detection of more than 1000 times of repeated variation is realized. The invention also provides an application of the PCR detection kit for the CTG region of the atrophic myotonic protein kinase gene. The PCR detection kit provided by the invention can be used for amplifying a hybrid allele sample carrying CTG repeated expansion, and can be used for detecting the number of CTG tribasic group repetition times of an atrophic myotonic protein kinase gene 3 '-UTR at the same time.

Description

technical field [0001] The invention belongs to the technical field of PCR amplification, and in particular relates to a PCR detection kit for the CTG region of atrophic myotonic kinase gene and its application. Background technique [0002] Myotonic Dystrophy Type 1 (DM1) is a hereditary disease characterized by muscle weakness, rigidity, and muscle atrophy, as well as endocrine, cardiac, ocular lens and other systems damage. It is currently known that DM1 is caused by the expansion of the CTG three-base repeat in the 3'-UTR region of the DMPK gene, and the number of CTG repeats in the DMPK gene varies from 5 to 5000 times. [0003] There are many PCR amplification methods for the determination of CTG repeats in the DMPK gene, such as triple-primer PCR (TP-PCR), heat pulse PCR (HPE-PCR), Small-pool-PCR (SP-PCR), and Flash-small -pool PCR (FSP-PCR). Compared with non-amplified southern-blot and third-generation sequencing, these methods have the advantages of less sample ...

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Problems solved by technology

However, the PCR amplification method itself is affected by factors such as the structure of the primer itself, the degree of firmness with the template, the size of the target gene fragment, etc., resulting in significant differences in the results.

In most cases, the number of CTG repeats in the two alleles of DMPK in DM1 patients is one large and one small, and most of the currently known primers have a bias, that is, they tend to amplify small fragments, resulting in large fragments. Difficult to detect

[0004] At present, TP-PCR is a relatively mature technology, but it can only detect the definite value of the number of CTG repetitions within 240 times. For the case of a large number of repetitions (>240 repetitions), it cannot accurately give the number of repetitions (such as China A set of primers and detection kits for detecting CTG repeat sequences disclosed in patent document 201410211499.0)

In addition, there have been reports in recent years (Musova Z, Mazanec R, Krepelova A, et al.Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene[J].2009,149A(7):1365-1374.), TP -Although the PCR method has high sensitivity and specificity, about 5% of the false negative samples may be caused by: 1) interference sequences such as CCG / CTC and GGC exist in the repeated sequence; There are mutations in the binding region of some samples; 3) some accidental factors in the amplification process

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