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Primer composition for detecting fragmentized DNA target area and application thereof

A technology of primer composition and target region, which is applied in the field of primer composition for detection of fragmented DNA target region, can solve the problems of low mixing ratio, need for improvement, detection method is difficult to achieve such a high detection sensitivity, etc., to improve the scope of application , The sequencing results are accurate and reliable, and the effect of improving the scope of adaptation and the amount of effective templates

Pending Publication Date: 2016-10-12
GENETALKS BIO TECH CHANGSHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the different environments in which criminal investigation samples are located, the DNA of criminal investigation samples usually degrades to varying degrees
At the same time, the DNA of criminal investigation samples is often not pure DNA of a single individual, but a mixture mixed with other individual DNA, and the mixing ratio of truly meaningful DNA may be extremely low (0.1%). However, existing detection methods are difficult To achieve such a high detection sensitivity
[0006] Therefore, the current fragmented DNA detection method still needs to be improved

Method used

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  • Primer composition for detecting fragmentized DNA target area and application thereof
  • Primer composition for detecting fragmentized DNA target area and application thereof
  • Primer composition for detecting fragmentized DNA target area and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] 1. Connector design

[0134] Prelib-ADT-S: CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (I is the index area),

[0135] Prelib-ADT-AS: pGATCGGAAGAGC,

[0136] The above sequences need to anneal into double strands.

[0137] The structure of the product after connection:

[0138] Top: 5'-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT---AGATCGGAAGAGC-3',

[0139] Bottom: 5'-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT---AGATCGGAAGAGC-3',

[0140] Co-extension-specific primers were designed for exon 19 of EGFR.

[0141] The sequence of exon 19 of EGFR is as follows:

[0142] GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGAT (SEQ ID NO: 11),

[0143] The sequences of exon 19 of EGFR and its upstream and downstream intron regions are as follows:

[0144] CAGCCCCCAGCAATATCAGCCTTAGGTGCGGCTCCACAGCCCCAGTGTCCCTCACCTTCGGGGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTG...

Embodiment 2

[0221] 1. Connector design

[0222] Prelib-ADT-S: CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (I is the index area),

[0223] Prelib-ADT-AS: pGATCGGAAGAGC,

[0224] The above sequences need to anneal into double strands.

[0225] The structure of the product after connection:

[0226] Top: 5'-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT---AGATCGGAAGAGC-3',

[0227] Bottom: 5'-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT---AGATCGGAAGAGC-3',

[0228] Direct extension-specific primers were designed for the T790M point mutation in exon 20 of EGFR.

[0229] The sequence of exon 20 of EGFR is as follows:

[0230] GAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCTCCTGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAG (SEQ ID NO: 13),

[0231] The sequences of exon 20 of EGFR and its upstream and downstream intron regions are as follow...

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Abstract

The invention discloses a primer composition for detecting fragmented DNA target regions and applications thereof, wherein the primer composition includes: a first primer set, the first primer set includes a first universal primer and a second universal primer, the The target points of the first universal primer and the second universal primer are located at both ends of the target region, and are respectively used to constitute the 5' end and the 3' end of the sequencing library; and the second primer set, the second primer The sets include a first set of specific primers and a second set of specific primers. Using the primer composition of the present invention to amplify and enrich the target region of fragmented DNA can significantly improve the scope of primer amplification and the amount of effective template, and then perform sequencing detection on the enriched product, which can significantly improve the fragmented DNA. Detection sensitivity.

Description

technical field [0001] The invention relates to the technical field of fragmented DNA detection, in particular to a primer composition for detecting a fragmented DNA target region and an application thereof. Background technique [0002] When scientific researchers conduct biological research, they need to extract nucleic acid (DNA, or RNA) from various types of samples. Wherein, the body fluid sample contains fragmented DNA, and biological detection of the DNA in the sample is an important detection step. Plasma DNA, for example, is extracellular DNA in the blood and exists in the form of nucleosomes (DNA-protein complexes). The length of such fragmented DNA is tens to hundreds of base pairs (the main peak is 166bp). This kind of fragmented DNA is usually released into the blood circulation system by a small number of apoptotic cells, the content is extremely low, and it is difficult to detect reliable information by existing PCR methods. [0003] In addition, fossils ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B50/06C12M1/00
CPCC12Q1/6876C12Q1/6869C40B50/06C12Q2531/113C12Q2535/122
Inventor 王永利宋卓袁梦兮
Owner GENETALKS BIO TECH CHANGSHA CO LTD
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