Method and kit for detecting fragile X syndrome mutation

Abstract

The invention relates to a method and a kit for detecting fragile X syndrome mutation. The kit comprises the following reagents: (1) a reagent for long fragment PCR amplification; (2) a reagent for PCR amplification of a high GC region; and (3) a reagent for constructing a third-generation sequencing library. The method comprises the following steps: (1) obtaining a sample of a subject; (2) carrying out long fragment PCR (Polymerase Chain Reaction) amplification and high GC PCR amplification on the sample; (3) constructing a third-generation sequencing library; and (4) sequencing and analyzing the FMR1 gene mutation type.

Description

technical field [0001] The invention relates to a primer and a method for detecting multiple mutations of FXS by using a three-generation long-read sequencing platform, and a kit suitable for the method. Background technique [0002] Fragile X syndrome (FXS) is an X-linked incomplete penetrance genetic disease, and it is also one of the common chromosomal diseases. It is named after the brittle part that shows like a fracture. [0003] FXS is the most common cause of inherited intellectual disability, with an incidence of 1 / 4000 in males and a carrier rate of 1 / 8000-1 / 4000 in females. In most cases, FXS is caused by expansion of trinucleotide (CGG) repeats in the 5'-UTR region of the FMR1 gene on the X chromosome (Crawford DC et al. Genetics In Medicine, 2001, 3(5): 359-371) . Trinucleotide (CGG) can be divided into pre-mutation (55-200) and complete mutation (>200) according to the number of repeats; pre-mutation is unmethylated and can produce FMR1 protein with sligh...

AI Technical Summary

Problems solved by technology

[0008] 1. It is impossible to simultaneously detect all mutation types in the same system;

[0009] 2. Since there may be a certain proportion of chimeras in the CGG repeat expansion of FXS, CE and Southern blot may miss some low proportion chimeras;

[0010] 3. CE and Southern blot can only detect CGG repeats greater than 200, and cannot determine a higher number of CGG repeats;

[0011] 4. The judgment of CE on the number of CGG repeats less than 200 depends on the standard with known results, and it is impossible to directly and accurately judge the number of CGG repeats;

[0012] 5. Unable to accurately determine the type of AGG insertion in CGG repeats;

[0013] 6. MLPA cannot determine the exact location of the large fragment deletion;

[0014] 7. Current methods cannot directly detect microdeletions near unknown CGG repeat regions

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