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A method and reagents for the ultrasensitive detection of trace amounts of DNA from multiple pools of DNA

A sensitive detection and trace technology, applied in the field of DNA detection, can solve the problems of low detection sensitivity, achieve the effect of solving low detection sensitivity, increasing copy number, and increasing utilization rate

Active Publication Date: 2018-11-30
GENETALKS BIO TECH CHANGSHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention provides a method and reagents for ultrasensitive detection of trace DNA from various mixed DNAs, which solves the technical problem of low detection sensitivity of existing detection technologies for complex samples containing a large amount of foreign DNA contamination

Method used

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  • A method and reagents for the ultrasensitive detection of trace amounts of DNA from multiple pools of DNA
  • A method and reagents for the ultrasensitive detection of trace amounts of DNA from multiple pools of DNA
  • A method and reagents for the ultrasensitive detection of trace amounts of DNA from multiple pools of DNA

Examples

Experimental program
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Embodiment

[0037]In this example, library construction was performed on samples and seven sites of four genes NRAS, KRAS, PI3KA, and EGFR were detected, as follows:

[0038] 1. Adapter Design Containing Random Single Molecular Tags

[0039] IDX1-S:

[0040] CAAGCAGAAGACGGCATACGAGATNNNNNNNggaattaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 1);

[0041] IDX2-S:

[0042] CAAGCAGAAGACGGCATACGAGATNNNNNNNNatccggcGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 2);

[0043] IDX3-S:

[0044] CAAGCAGAAGACGGCATACGAGATNNNNNNNNcaggccgGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 3);

[0045] ADT-AS: pGATCGGAAGAGC (SEQ ID NO: 4); the phosphate group at the 5' end of the ADT-AS sequence is modified.

[0046] IDX1-S, IDX2-S, and IDX3-S (both are the first strands of the linker) are annealed with the ADT-AS sequence to form double strands respectively, forming three linkers ADT1, ADT2, and ADT3.

[0047] 2. This example detects seven common mutation points in the four genes of NRAS, KRAS, PIK3CA,...

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Abstract

The invention discloses a method and a reagent for ultra-sensitively detecting trace DNA (Deoxyribonucleic Acid) from multiple mixed DNAs. The method comprises the following steps of (a), using a probe to carry out probe preenrichment on a joint ligation product containing target DNA of a to-be-detected site or a to-be-detected area, wherein the probe is a double stranded oligonucleotide; joints at two ends of the joint ligation product both comprise binding sites of universal primers; (b), using an upstream specific primer and a downstream specific primer which aim at the to-be-detected site or the to-be-detected area and the universal primers at the two ends to carry out specific enrichment on a probe-preenrichment product, and then carrying out universal primer amplification on a specific-enrichment product. By using the method, the technical problem that an existing detection technique is low in detection sensitivity aiming at a complicated sample containing much foreign DNA pollution is solved.

Description

technical field [0001] The invention relates to the technical field of DNA detection, in particular to a method and a reagent for ultrasensitive detection of trace DNA from various mixed DNAs. Background technique [0002] Non-invasive sampling is often required for genetic testing in the fields of scientific research, clinical or health examination, that is, by obtaining human hair, nails, feces (containing intestinal exfoliated cells), urine, saliva, food residue (containing oral Exfoliated cells), vomit (containing exfoliated cells of the digestive tract), tissues or body fluids with moldy bacteria, etc. to detect specific DNA regions of a species (usually human); There have been instances where samples were poorly preserved or sampled under specific circumstances. These sample components are often extremely complex. DNA extracted from these samples often contained a large amount of DNA from non-target species. For example, the DNA extracted from a stool sample is a mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2565/525C12Q2525/191
Inventor 王永利宋卓袁梦兮
Owner GENETALKS BIO TECH CHANGSHA CO LTD