Novel capture and enrichment technology for targeting nucleic acid molecules

A technology targeting nucleic acids and molecules, which is applied in the field of capture and enrichment of nucleic acid molecules, can solve the problems of high probe labeling price, high capture cost, connection efficiency affecting capture and enrichment yield, etc., to improve detection sensitivity, Improve hybridization efficiency and increase the effect of collision chance

Inactive Publication Date: 2014-02-26
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since this method requires one-step covalent connection, the connection efficiency directly affects the yi

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  • Novel capture and enrichment technology for targeting nucleic acid molecules

Examples

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Embodiment 1

[0044] Example 1 Using polyacrylamide gel capture technology and high-throughput sequencer to analyze the distribution of various functional genes in the intestinal microbial metagenome:

[0045] (1) Design and preparation of probes

[0046] Selection of DNA sequence fragments of functional genes of intestinal microorganisms: use software such as Mega or Clustal W to perform bioinformatics comparison and complete the selection of 20,000 functional gene nucleic acid sequences.

[0047] Probe design: use software such as primer 5.0 to design probes for specific fragments of microorganisms.

[0048] Synthesis and labeling of probes: Probes were synthesized using a DNA synthesizer, and after all probes were synthesized, an acrylamide group was labeled at the 5' end.

[0049] (2) Intestinal microbial community metagenomic extraction, genome fragmentation and adapters on both sides

[0050] Intestinal microbial community metagenomic DNA extraction: collect 200mg of fresh stool, ...

Embodiment 2

[0064] Example 2 Targeted analysis of exon mutations in cancer-related genes in the human genome using polyacrylamide gel capture and high-throughput sequencing:

[0065] (1) Design and preparation of probes

[0066] Exon nucleic acid sequence selection of cancer-related genes: use software such as Mega or Clustal W to perform bioinformatics comparison and complete the selection of 1,000 functional gene nucleic acid sequences.

[0067] Design of probes: use primer5.0 and other software to design probes for specific fragments of microorganisms.

[0068] Synthesis and labeling of probes: Probes were synthesized using a DNA synthesizer, and after all probes were synthesized, an acrylamide group was labeled at the 5' end.

[0069] (2) Intestinal microbial community metagenomic extraction, genome fragmentation and adapters on both sides

[0070] Human genomic DNA extraction: Collect 2ml of venous blood, lyse red blood cells and digest with proteinase K, and extract human genomi...

Embodiment 3

[0084] Example 3 Targeted analysis of trace pathogenic microorganisms in food using polyacrylamide gel capture and high-throughput sequencer:

[0085] (1) Design and preparation of probes

[0086] Species-specific DNA sequence fragment selection: use software such as Mega or Clustal W to perform bioinformatics comparison and complete the selection of more than 100 specific genes of food pathogenic microorganisms.

[0087] Probe design: use software such as primer 5.0 to design probes for specific fragments of microorganisms.

[0088] Synthesis and labeling of probes: The probes were synthesized using a DNA synthesizer, and an acrylamide group was labeled at the 5' end.

[0089] (2) Genome extraction of pathogenic microorganisms, genome fragmentation and joints on both sides

[0090] Genomic DNA extraction of pathogenic microorganisms: collect chilled meat and duck meat products after slaughtering and processing, and use sterile cotton swabs to conduct six-sided 50cm 2 Af...

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Abstract

The invention discloses a novel capture and enrichment technology for targeting nucleic acid molecules. The targeting nucleic acid molecule fragments are hybridized through special capture probes; and probe molecules marked with acrylamide groups are fixed in acrylamide glue, so that the targeting nucleic acid molecule fragments are captured. The technology can be used for functional gene capture and high-throughput sequencing analysis of microbial community, fishing and high-throughput sequencing analysis of genes related to diseases in human genome, and fishing and analysis of signature sequences of environment pathogenic microorganisms. Besides, the technology can be used for development of diagnostic kit for a plurality of molecular diseases and pathogenic microorganisms. The technology is relatively low in cost. Glue efficiency of acrylamide molecules is far higher than connection efficiency of magnetic beads and probes, so that capture efficiency is greatly increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a technique for capturing and enriching nucleic acid molecules. Background technique [0002] The emergence of a new generation of high-throughput sequencers has greatly reduced the cost of nucleic acid sequencing. Using high-throughput sequencers, a large number of nucleic acid sequences have been sequenced in just a few years. The entire genomes of various organisms have been sequenced, and many personal genomes have also been sequenced. Human whole genome sequencing is an important application of high-throughput sequencers. However, most research and diagnostic goals only require sequencing a specific fragment in a large number of samples. For example, for the human genome, exons are protein-coding regions, and exons only make up about 1% of the whole genome. In most cases, especially for the screening of disease-associated exon mutations, only the exon region is sufficient. In...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 周东蕊张红琳章爱娣肖鹏峰白志茂陆祖宏
Owner SOUTHEAST UNIV
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