Method for measuring nucleotide sequence of disease associated nucleic acid molecules in sample to be detected

A nucleotide sequence and nucleic acid molecule technology, applied in the field of nucleotide sequence determination of disease-related nucleic acid molecules in samples to be tested, can solve problems such as inability to interpret mutation sites, high sequencing costs, and inability to simultaneously detect single-gene diseases.

Active Publication Date: 2012-01-25
BGI GENOMICS CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

There are many shortcomings in the above methods, such as: pedigree analysis, chromosome karyotype analysis, enzymatic reaction activity determination method and FISH method are all detection at the chromosome level, and the accuracy is low; RALF, SSCP and MOLDI-TOF analysis methods are Indirect detection methods cannot directly reflect changes in sites; a-CGH, ​​qPCR, and MLPA can only target specific sites and cannot interpret newly discovered mutation sites. PCR amplification process
Therefore, although the first-generation sequencing technology based on the Sanger method is currently the gold standard for the detection of monogenic diseases, due to the small number of samples sequenced at the same time, the types of monogenic diseases detected are limited to one or several, The cost of sequencing is high, and it is impossible to detect multiple single-gene diseases with known molecular basis at the same time, which greatly limits the identification of individual genetic diseases
[0005] At present, there is no effective method for determining the nucleotide sequence of the disease-associated nucleic acid molecule in the sample to be tested

Method used

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  • Method for measuring nucleotide sequence of disease associated nucleic acid molecules in sample to be detected

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preparation example Construction

[0079] The preparation method of the library is well known to those skilled in the art, including (but not limited to) steps:

[0080] 1. A sample to be detected is provided, the sample contains an interrupted double-stranded nucleic acid fragment derived from genomic DNA, and the nucleic acid fragment has a blunt end;

[0081] 2. For the double-stranded nucleic acid fragment in the previous step, add an adapter connection sequence at the end; and through the adapter connection sequence, add an adapter at both ends of the double-stranded nucleic acid fragment, wherein the adapter has a primer binding region and a connecting complementary region, the connecting complementary region is complementary to the linking sequence of the linker; the sequences of the primer binding regions of the linkers at the 3' end and the 5' end of both sides are different.

[0082] 3. Amplify the DNA double-stranded nucleic acid fragment with the adapter obtained in the previous step with the first ...

Embodiment 1

[0142] Establishment of chip hybridization platform

[0143] The probes are designed from the exon sequence and 100 bp before and after the exon of the known disease-causing gene of single gene disease, a total of more than 70,000 probes, its SEQ ID NO., chromosomal coordinates, capture position, length and the disease involved See Table 4 for the species.

[0144] Table 4

[0145]

Embodiment 2

[0147] DNA library preparation

[0148] 1. Genomic DNA acquisition

[0149] Take human peripheral blood, extract genomic DNA, and obtain 3 μg DNA.

[0150] 2. DNA Fragmentation

[0151] The extracted human genomic DNA sample is fragmented on a Covaris S2 instrument (purchased from Covaris, USA), and finally broken into a mixture of DNA double-stranded fragments with a main band of 200bp, and the fragments are purified. The purification process Adopt Ampure Beads method, carry out according to Agencourt AMPure protocol (U.S. Beckman Company).

[0152] 3. DNA fragment ligation

[0153] The DNA fragments are end-repaired to become a fragment mixture with blunt ends, and an "A" is added to the 3' end of each single strand to facilitate connection with adapters with "T". After connection, purify and purify Methods Ampure Beads were used according to the Agencourt AMPure protocol (Beckman Company, USA). After purification, excess reagents such as buffer, enzyme, ATP, etc. are r...

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Abstract

The invention relates to a method for measuring a nucleotide sequence of disease associated nucleic acid molecules in a sample to be detected. The method comprises the following steps of: adding joints to the terminals of double-chain nucleic acid molecules fragmented in the sample to be detected and from genome DNA, and performing enrichment; and capturing the DNA fragments containing joints by using a nucleic acid chip, and sequencing the captured fragments on a high-flux sequencing platform. The nucleotide sequence of the disease associated nucleic acid molecules in the sample can be quickly obtained with high flux by analyzing the sequencing result based on the known gene locus information, and the nucleotide sequence can be used for detecting monogenic disease. The invention also provides the nucleic acid chip used for the method and fixed with several kinds to tens of thousands of kinds of disease specific probes, and a kit containing the chip.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for determining the nucleotide sequence of a disease-related nucleic acid molecule in a sample to be detected. The method includes: designing a chip with multiple disease-specific probes, capturing and enriching specific target DNA fragments with adapters, high-throughput sequencing, and analyzing gene mutation information. Background technique [0002] The completion of genome sequencing of various model organisms has greatly improved people's understanding of the pathogenic mechanism of diseases and the physiological state of the body at the genetic level, and has also greatly promoted the development of second-generation high-throughput sequencing technology. Organisms whose genomes have been sequenced so far include: human, mouse, rat, fruit fly, rice, soybean, Arabidopsis, etc. However, due to the limitation of sequencing cost, genome sequencing of individuals and iden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/68C12Q1/6883C12Q1/6874C12Q1/6869C12Q2600/156
Inventor 魏晓明陈洋杨光辉朱倩谢姝琦汪建王俊杨焕明
Owner BGI GENOMICS CO LTD
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