Maple syrup urine disease related gene mutation, and detection method and purpose thereof

A gene, α subunit technology, applied in the field of disease-related mutant genes, can solve problems such as loss, decreased branched-chain α-keto acid decarboxylase activity, etc.

Active Publication Date: 2014-10-15
BGI GENOMICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the E1α subunit reduce or completely abolish branched-chain α-ketoacid decarboxylase activity

Method used

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  • Maple syrup urine disease related gene mutation, and detection method and purpose thereof
  • Maple syrup urine disease related gene mutation, and detection method and purpose thereof
  • Maple syrup urine disease related gene mutation, and detection method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] genome extraction

[0068] Peripheral blood was collected from all subjects, and genomic DNA was extracted using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer.

Embodiment 2

[0070] Chip design, library construction, and high-throughput sequencing

[0071] The inventors designed a capture chip (NimblGen, Roche) to capture the exons, splice sites and adjacent intron sequences of the four genes BCKDHA, BCKDHB, DBT and DLD.

[0072] For the method of library construction, please refer to the protocol provided by Illumina (http: / / www.illumina.com / ). The brief description is as follows: Genomic DNA was randomly broken into fragments of 200-300 bp using an ultrasonic instrument (Covaris S2, Massachusetts, USA). Take 1 μg (quantified by Nanodrop) of purified DNA fragments and treat them with T4 DNA polymerase, T4 phosphonucleotide kinase and Klenow fragment of Escherichia coli DNA polymerase to fill in the 5'overhanging ends and remove the 3'overhanging ends. A base is added to the 3' end and then a linker sequence is ligated. After adding adapters, PCR primers containing 8 bp tag sequence (barcode sequence) were used to perform 4 cycles of PCR to obtai...

Embodiment 3

[0074] Variant detection, annotation and comparison with databases

[0075] 1) After the sequencing is completed, use Illumina1.3.4 for image analysis, error rate estimation and base reading to obtain raw data. Low-quality reads (reads) and reads containing adapter contamination are then filtered. We will use BWA to align high-quality reads to the reference genome for reads containing more than 10% of N bases and 50% of the base-occupied reads with a quality value of less than 5 to obtain the only alignment on the genome Read paragraph. Then use SOAPsnp and GATK to detect SNP and indel respectively.

[0076] 2) For the detected SNP and indel variants, we use the internal process to annotate, that is, to determine the genomic environment of the variant and the type of the variant. We focused on non-synonymous mutations, splice acceptor / donor site mutations, and coding region insertion and deletion mutations, the three most likely disease-associated mutations. We compared th...

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Abstract

The invention relates to the field of disease related gene mutation, especially gene mutation of autosomal recessive hereditary amino acid metabolism disease, maple syrup urine disease related gene mutation, and more particularly maple syrup urine disease related gene mutation, and a detection method and purpose thereof.

Description

technical field [0001] The invention relates to the field of disease-related mutation genes, in particular to autosomal recessive genetic amino acid metabolism disease gene mutation and maple diabetes related gene mutation. Background technique [0002] Maple Syrup Urine Disease (MSUD) is an autosomal recessive genetic disorder of amino acid metabolism, mainly due to the following reasons: the gene defect of the branched-chain amino acid α-ketoacid dehydrogenase complex makes the branched-chain Amino acids (mainly including leucine, isoleucine and valine) after the transamination reaction form corresponding branched-chain α-keto acids cannot be oxidatively decarboxylated, branched-chain amino acids and branched-chain α-keto acids in tissues are abnormally increased, and body fluids It excretes alpha-keto acids, giving it a maple syrup-like smell, and hence its name. At the same time, because the branched-chain amino acids and their α-keto acid derivatives accumulated in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12Q1/68C12N15/11
Inventor 魏晓明朱倩
Owner BGI GENOMICS CO LTD
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