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Method for assessing base damage, mismatch and variation in sample DNA by using first-generation sequencing

A base and sample technology, applied in the field of first-generation sequencing to evaluate base damage, mismatch and variation in sample DNA, can solve problems such as long time period, unfavorable detection project promotion, molecular labeling technology dependence, etc., to achieve optimized preservation methods, Optimizing the performance of extraction techniques

Active Publication Date: 2022-03-08
CARRIER GENE TECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in terms of technology, if you want to detect a low proportion of mutation information, you must be inseparable from molecular labeling technology, but molecular labeling technology relies heavily on high-depth sequencing, and the time period is long, which is not conducive to the promotion of detection projects

Method used

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  • Method for assessing base damage, mismatch and variation in sample DNA by using first-generation sequencing
  • Method for assessing base damage, mismatch and variation in sample DNA by using first-generation sequencing
  • Method for assessing base damage, mismatch and variation in sample DNA by using first-generation sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: Using next-generation sequencing to assess the degree of base damage in sample DNA

[0096] 1. Set the sampling area at 4 positions of the human genome, and design the combination of primer pairs for PCR, as shown in the following table:

[0097] name Seq(5'-3')(SEQ ID NO.1~13) 50mM, 25°C, deltaG DmDe1-FP CCCTGACAACATAGTTGGAATCA -27.4 DmDe1-RP ACTCCAGGATAATACACATCACAGT -29.2 DmDe1-BL TGGAATCACTCATGATATCTCGAGCCAT -34.0 DmDe2-FP AGCAGTCTCTGCCTCGC -24.5 DmDe2-RP AGAAGATTCGGCAGAACTAAGCA -28.5 DmDe2-BL CCTCGCCAAGCGGCTCATGTTAATATT -35.0 DmDe4-FP AGAAGATGTGGAAAAGTCCCAATG -28.4 DmDe4-RP GTGCCCAGGTCAGTGGAT -24.7 DmDe4-BL TCCCAATGGAACTATCCGGAACATCCA -34.1 DmDe6-FP TCCTTTAACCACATAATTAGAATCATTCTTGA -33.9 DmDe6-RP AGTTAGTTTTCACTCTTTACAAGTTAAAATGA -33.5 DmDe6-BL ATCATTCTTGATGTCTCTGGCTAGACCAAAA -35.6 UNITag tgtaaaacgacggccagtaca

[0098] Note: the RP seq...

Embodiment 2

[0122] Example 2: Logical demonstration of analyzing the number of molecular labels UMInum from sanger results

[0123] 1. Prepare 100 kinds of molecular tags with known sequences, each molecular tag is 28nt, and each base is separately occupied, such as figure 2 shown.

[0124] 2. Assume that the PCR product contains such image 3 The five molecular tag sequences shown, after generation sequencing, according to the sanger results, at each position N - information is available, such as image 3 shown.

[0125] 3. According to N - For the known sequence of information filtering molecular tags, for example, at the 16th base, it is necessary to exclude molecular tags that do not contain g and t at this position. - After the elimination of information, only 15 kinds of molecular labels are left, such as Figure 4 shown;

[0126] 4. Continue according to N - Information is excluded, and when the 28th base is reached, 5 molecular tags are finally left, which happen to be th...

Embodiment 3

[0129] In order to illustrate that the scheme of the present invention has more general practicability, this example is designed at other human genome positions different from Example 1, which is the same as the principle of primer design in Example 1. The primer design principle of this example refers to the company's earlier CN110923325A "Primer Blocker Set, Kit and Method for Detecting EGFR Gene Mutation" and CN110982884A "Primer Set, Kit and Method for Detecting AML-related Gene Mutation";

[0130] SSL3-FP: CCAGAAAACAGGCAGGTCTCTC

[0131] SSL3-BL: CAGGTCTCTCTGCTCTTGACCGAGC

[0132] SSL3-RP: ACAGCAGGCAGTTGGGA

[0133] The UNITaq sequence is the same as in Example 1, and the SSL3-RP sequence in this example is only a specific sequence part. During the preparation process, the UNITaq sequence needs to be added to construct the 5-tgtaaaacgacggccagtaca(N28)-RP structure, wherein N28 is as follows Figure 6a and 6b For the 100 UMI sequences shown, the design of UMI is partly ...

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Abstract

The invention relates to a method for evaluating base damage, mismatch and variation in sample DNA (deoxyribonucleic acid) by using first-generation sequencing, which comprises the following steps: in a PCR (polymerase chain reaction) amplification process, on one hand, marking original DNA molecules with damage or mismatch by using a molecular tag, and on the other hand, carrying out enrichment amplification amplification on a sampling region, amplifying about 0.1% of damage or mismatch information to 10-99%, and then respectively evaluating proportion values of base damage, mismatch and variation in the sample DNA by adopting an evaluation method based on an enrichment and amplification effect and an evaluation method based on the number of molecular tag types, and judging the proportion values of base damage, mismatch and variation in the sample DNA according to credible results of the two methods. According to the method disclosed by the invention, the true existence of damage or mismatch can be accurately confirmed by adopting an economical and rapid sanger sequencing method, the sample DNA extraction technology and the preservation method can be favorably optimized, and the quality of the sample DNA is helped to be evaluated.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a method for evaluating base damage, mismatch and variation in sample DNA by generation sequencing. Background technique [0002] With the development of technology, in the field of DNA detection, especially in the field of cancer detection, people are paying more and more attention to the low proportion of mutation information. For example, 0.1% somatic mutation information is one of the key indicators in the current liquid biopsy field. Gradually, People will no longer be satisfied with the index of 0.1%, but require further progress. If it reaches the level of 0.01%, they will face the problem of how to distinguish mutations from mismatches and base damage. [0003] First, clarify the meaning of the two concepts of mutation and mismatch. At the level of single-copy cells, such as single sperm and eggs, they are haploid. The concept of mutation is difficult ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869G16B25/20G16B30/00G16B50/30
CPCC12Q1/6869G16B25/20G16B30/00G16B50/30C12Q2531/113C12Q2535/101
Inventor 罗俊峰王一帆徐雪陈曦宋萍
Owner CARRIER GENE TECH SUZHOU CO LTD