Method and kit for sequencing phenylalanine hydroxylase gene

Abstract

The invention relates to the field of genetic engineering, and provides a method and a kit for sequencing a phenylalanine hydroxylase (PAH) gene. The method comprises the following steps: A. amplifying a plurality of target regions in a sample to be detected by using a PAH gene specific primer, and constructing a sequencing library based on an amplified product; B. conducting single-molecule amplification for the sequencing library to obtain a plurality of single-molecule amplified products corresponding to the plurality of target regions; and C. conducting high-throughput gene sequencing for the plurality of single-molecule amplified products to obtain the sequence information of the plurality of target regions. By utilizing the method and the kit, the plurality of target regions are simultaneously sequenced by using the high-throughput gene sequencing technology so that the detection efficiency is improved, the accuracy and sensitivity of detection can be also improved, and furthermore, multi-region detection can be conducted for a great deal of samples simultaneously.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method and kit for sequencing the phenylalanine hydroxylase gene. Background technique [0002] Phenylalanine hydroxylase (phenylalanine hydroxylase, PAH) gene is located on chromosome 12 (12q22-12q24.1), consisting of about 1.5Mb bases, the coding region contains 13 exons and 12 introns, mRNA The size is 1353bp, translated into an enzyme monomer containing 451 amino acids. The mutation of PAH gene has the following characteristics: 1. The position of mutation is variable; 2. The type of mutation is diverse. So far, more than 530 kinds of PAH gene mutation types have been found, covering all exons, and more than 30 kinds have been found in China, of which more than 60% are missense mutations. PAH is an amino acid metabolizing enzyme produced by the liver, which catalyzes the reaction of phenylalanine into tyrosine, thereby participating in the process of gluconeogenesis, an...

AI Technical Summary

Problems solved by technology

[0003] At present, the common method for sequencing the PAH gene is Sanger sequencing, which can detect the target region, but the Sanger sequencing method can only detect a certain region of the PAH gene (not greater than 900bp) in one sample at a time. Sequencing, the detection efficiency is low, and the cost of sequencing is high. Due to the limitation of the sanger sequencing principle, double peaks or even multiple peaks will appear when detecting the signal of samples with heterozygotes, resulting in disordered sequencing peaks obtained by sequencing, and even cannot be analyzed. Sequence information obtained, sequencing failed

A large number of studies have confirmed that the direct sequencing method of PCR products can only detect templates with a ratio of ≥20% in the mixed templates, and cannot obtain the precise variation ratio of the mutation site

And there is same defect in the kit based on Sanger sequencing method in the prior art

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