Screening method of molecular reverse probes for capturing specific region of enriched genome

A molecular reverse probe, a specific region technology, applied in the field of genetic engineering probes, which can solve the problems of the optional range of probes and the less consideration of experimental characteristics.

Active Publication Date: 2019-07-16
AMOY DIAGNOSTICS CO LTD
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The traditional design method mainly considers the specificity of the primers at both ends to select,

Method used

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  • Screening method of molecular reverse probes for capturing specific region of enriched genome
  • Screening method of molecular reverse probes for capturing specific region of enriched genome
  • Screening method of molecular reverse probes for capturing specific region of enriched genome

Examples

Experimental program
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Embodiment 1

[0075] Embodiment 1: detect the MIP probe design of human gene BRCA1 / 2 exon region

[0076] Using the method of the present invention, the molecular reverse probe design of the single-molecule tag marking is carried out on the coding region and the cut region of the BRCA1 and BRCA2 genes in the human genome. The coverage of this probe design for targeted capture was counted, and some of the probes were experimentally verified, and the uniformity and specificity of the sequencing data were systematically evaluated.

[0077] The specific design process is as follows:

[0078] 1. Selection of probe structure:

[0079] In this experimental design, the expected detection target is 1% of Somatic mutations, so UIDs are introduced into the probe structure, and the UIDs are distributed on both sides of the linker region. 6bp random bases were used each time, and the 2bp near the tether or extension arm were designed as random bases mutually exclusive with the reference genotype.

[...

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Abstract

The invention discloses a screening method of molecular reverse probes for capturing a specific region of an enriched genome. The method comprises the steps that firstly the structures of the molecular reverse probes are designed; starting from the 5' end of the target region, the probes with at least 50% of scanned areas falling on the 5' end are selected to serve as a candidate starting set; adjacent probes are scored in pairs, the probes meeting the requirement are combined with the previous selected probes sequentially to form a set of all possible probe combinations, each candidate combination is scored, score calculation is conducted on all possible combinations, and the combination with highest scores or lowest penalty scores is obtained. The method is applicable to linker structures of different sequencing platforms, an obtained molecular reverse probe technique is remarkably improved at the aspects of precision, convenience, detection sensitivity and the like, meanwhile, a coverage target region, especially the number of the probes needed by a klenow fragment exon region is effectively reduced, and the method is convenient and rapid, and saves the cost.

Description

technical field [0001] The invention relates to the field of genetic engineering probes, in particular to a screening method for molecular reverse probes used to capture and enrich specific regions of genomes. Background technique [0002] Rare mutations and de novo mutations are the genetic basis of complex diseases. It is of great significance to detect the genetic diversity in a sample cell population for the study of human cancer genetics. However, despite the decreasing cost and increasing throughput of massively parallel sequencing, it is still very challenging to stably detect mutations with low relative abundance in a specific DNA sample. [0003] Many methods have been used to identify gene mutations, including qPCR, mass spectrometry, microarray chips, etc. However, these methods are not very sensitive to the identification of low-frequency mutations. Therefore, there is an urgent need for a method that can handle a large number of DNA molecules and can more sensi...

Claims

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Application Information

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IPC IPC(8): G16B25/20
CPCC12Q1/68G16B25/00
Inventor 李旭超张昭金保雷张林华葛会娟陈宁阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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