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Construction method of whole-genome exon sequence capture probe

A sequence capture and genome-wide technology, applied in recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of limited species capture and high cost

Inactive Publication Date: 2015-10-07
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are commercial exon capture kits, the current capture species are limited, limited to humans and mice, and the cost is extremely expensive ($350 per sample) and other defects

Method used

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  • Construction method of whole-genome exon sequence capture probe
  • Construction method of whole-genome exon sequence capture probe
  • Construction method of whole-genome exon sequence capture probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. Extraction of total RNA from normal human blood

[0056] Subjects: normal people

[0057] Sample collection method: blood collection with anticoagulant tubes, and immediately put the blood collection tubes into the ice box

[0058] Experimental steps:

[0059] Ribonucleic acid quality inspection: use Trizol method to extract normal human blood, Trizol method for mRNA extraction is relatively mature, you can refer to textbooks such as Molecular Cloning Experiment Guide, or use existing commercially available kits, such as invitrogen; No.15596- 026 kit, perform gel electrophoresis detection on the extracted RNA (see Chapter 5 of the third edition of Molecular Cloning), and see the results in figure 1 .

[0060] 2. Capturing exon sequences

[0061] (1) Interrupting mRNA

[0062] Dilute 4ug of total RNA with 50 μL of nuclease-free ultrapure water, add purified magnetic beads (RNA Purification Beads 15026778illumina) at a volume ratio of 1:1, incubate at 65°C for 5 m...

Embodiment 2

[0121] Use the peripheral blood of patients with depression, use Trizol to extract blood RNA, and then conduct experiments according to the technical scheme of Example 1, construct exon capture reagents, and conduct experiments. The results are analyzed by bioinformatics, after removing joints and removing low-quality After the data filtering operation, high-quality sequences were obtained, compared with the reference genome through bwa and converted and calibrated using picard and GATK software, and statistics were performed, and it was known that the effective data we measured could cover more than 90% of the exons

[0122] More than 90% of exons were successfully covered.

[0123]

Embodiment 3

[0125] Using the peripheral blood of rats, Trizol was used to extract blood RNA, and then the above scheme was used to conduct experiments, to construct exon capture reagents, and to conduct experiments. The results were analyzed by bioinformatics, and after data filtering operations such as removing connectors and removing low-quality data , to obtain a high-quality sequence, compare it with the reference genome through bwa and use picard, GATK software to convert and calibrate, and then perform statistics. It is known that the effective data we measured can cover more than 90% of the exons.

[0126]

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Abstract

The invention relates to a kit for capturing a whole-genome exon sequence. The kit is a target sequence capture system based on the hybridization principle. An exon capture probe is designed, and a large quantity of exomes are obtained by hybridization to perform high-throughput sequencing and human whole genome comparison; and then, data analysis is performed, and the capture efficiency is calculated. The kit provided by the invention can provide high-quality target sequence enrichment, and has high specificity, high accuracy and excellent reproducibility in whole genome exon sequencing; the whole genome exon sequencing becomes the most effective policy for identifying the disease-causing gene of the Mendel disease; and the kit is also applied to the study and clinical diagnosis of the gene susceptible to complex diseases, and has a wide application prospect.

Description

technical field [0001] The present invention relates to the field of gene sequencing, in particular, to a target sequence capture system based on hybridization principle, and more specifically, to a method for constructing a whole-genome exon sequence capture probe. Background technique [0002] The development of next-generation sequencing technology has opened up a new situation for the research of modern genomics. However, the cost of whole genome sequencing and the complexity of analysis still make it difficult for researchers. The emergence of target sequence capture technology has alleviated these problems. Target sequence capture sequencing is sequencing performed after designing probes for known specific genomic regions. Exome capture sequencing is a special case, which is dedicated to the study of all exon regions in the genome. Whole-genome exon sequencing is a genomic analysis method that uses sequence capture technology to capture and enrich DNA in the exon regi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/11
Inventor 李明珍吴宁钱嘉林马媛媛武慧娟钱春燕李丽
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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