Amplification primer for detecting PAH (phenylalanine hydroxylase) gene mutation, kit and detection method thereof

Abstract

The invention discloses an amplification primer for detecting PAH (phenylalanine hydroxylase) gene mutation, a kit and a detection method thereof. A primer group for detecting PAH gene mutation through PCR (polymerase chain reaction) specific amplification comprises 9 pairs which are respectively primers used for amplifying PAH gene exon 1 upstream lateral sequences (positioned at the gene 5' end)to an introne 2 (a forward primer is shown as SEQ ID NO:1, and a reverse primer is shown as SEQ ID NO:2), primers are used for amplifying PAH gene introne 2 upstream (5' end) to introne 2 downstream(3' end) (a forward primer is shown as SEQ ID NO:3, and a reverse primer is shown as SEQ ID NO:4), and the like. The PAH gene whole coverage is realized; the detected sequence length reaches 88171bp;the intron variation and structure variation detection can be facilitated; the detection rate of pathogenic mutation can be improved; the operation is simpler; the cost is low; in addition, each amplification fragment and the adjacent fragment have the overlapping region; the primers can be used for fragment splicing and haplotype construction.

Description

technical field [0001] The invention relates to the technical field of PAH gene, in particular to an amplification primer, a kit and a detection method for detecting PAH gene mutation. Background technique [0002] Phenylalanine hydroxylase deficiency (PAHD) is a common amino acid metabolism disease. The pathogenesis of PAHD is due to the reduction or loss of enzyme activity caused by the mutation of phenylalanine hydroxylase (PAH), which makes the Abnormalities in phenylalanine metabolism. Untreated children with PAHD can cause severe intellectual disability and symptomatic epilepsy due to the neurotoxic effects of excess phenylalanine and bypass metabolites. If early diagnosis and treatment can be obtained, nerve damage may not occur and intelligence will be normal. With the development of clinical diagnosis and treatment, PAHD has become a treatable and preventable disease. Because children do not show symptoms in the early stage, PAH gene mutation analysis is the meth...

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Problems solved by technology

CN1335408A (2002) announced a special DNA chip for PAH gene mutation diagnosis suitable for early diagnosis and prenatal screening of phenylketonuria, but this method only detects 40 kinds of PAH gene mutations

CN1696308A (2005) also announced a special DNA chip for PAH gene mutation diagnosis suitable for early diagnosis and prenatal screening of phenylketonuria, but this method only detects 6 kinds of PAH gene mutations

CN101570787A (2009) also announced a compound chip for rapid prenatal diagnosis, but this method only detects 5 kinds of PAH gene mutations

CN201376967Y (2010) announced a compound chip for rapid prenatal diagnosis, but this method only detects 5 kinds of PAH gene mutations

CN103509865A (2014) announced a method for detecting PAH gene mutations using high-resolution melting curve analysis technology. It can mainly detect whether the sequences of exons 3, 6, 7, 11, and 12 of the PAH gene are abnormal, but cannot detect specific genotype

The total length of exons of PAH gene is 4122bp (the reference genome is GRCh37 / hg19), while the total length of introns is as high as 76597bp (the reference genome is GRCh37 / hg19). The above-mentioned reporting technology only detects mutation sites, or detects PAH The exon sequence of the gene and a small part of the intron sequence located at the flanks of the exon cannot be detected for the entire intron sequence of the PAH gene. There is still a need in the art for a gene detection method capable of detecting the full length of the PAH gene, namely This method can detect not only the entire exon sequence, but also the entire intron sequence

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