Amplification method for analyzing CGG repeat number of upstream untranslated region of fragile X mental retardation 1 (FMR1) gene

Abstract

The invention discloses an amplification method for analyzing a CGG repeat number of an upstream untranslated region of a fragile X mental retardation 1 (FMR1) gene, and belongs to the technical fieldof medical laboratory science. The amplification method comprises the following steps: (1) constructing three complexes that transcription activator-Like effectors (TALE) and deaminase are covalentlycoupled; (2) expressing the three complexes in escherichia coli; (3) testing the activity of proteins expressed by the three complexes; (4) extracting target DNA; (5) adding a reaction buffer and recombinant TALE-deaminase into the target DNA for reacting; (6) amplifying the DNA, after undergoing a deamination reaction, through a polymerase chain reaction (PCR); and (7) determining a sequence ofa PCR amplification product by using a Sangel sequencing method. According to the amplification method, TALE-deaminase is utilized to treat a sample, and a GC base in a specific region in the sample is directionally converted into an AT base, thereby reducing the GC content in the sample, so that isometric amplification and sequencing of the gene become feasible, and the CGG repeat number of the upstream untranslated region of the FMR1 gene is analyzed more accurately.

Description

technical field [0001] The invention belongs to the technical field of medical laboratory science, and specifically refers to an amplification method for analyzing the number of CGG repeats in the upstream untranslated region of the FMR1 gene. Background technique [0002] Fragile X syndrome (fragile X syndrome, FXS) is an X-linked genetic disease. Its typical features are moderate to severe mental retardation, giant testicles (50% of patients have a testicular volume of 30-50ml, and the normal value is 20ml), large ears, language barriers, mental retardation, and an IQ of 0-50. Most of the patients are men and the symptoms are severe in men. Females have two X chromosomes, most of them are carriers, 2 / 3 of them have normal intelligence, and 1 / 3 have mild mental retardation. In Fragile X syndrome families, diseases related to the etiology of Fragile X syndrome may appear, including Fragile X-associated tremor / ataxia syndrome (FXTAS), Fragile X-related primary Ovarian insu...

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Three-primer PCR (TRP-PCR) can increase the amount of amplified full mutations, but the amplified products

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