Application of single primer amplification library building technology in detection of fragmented rare DNA molecular mutation and kit

Abstract

The invention discloses application of a single-primer amplification library building technology in detection of fragmented rare DNA molecular mutation.The single-primer amplification library building technology comprises the following steps that target DNA is linearly amplified through a specific primer, a linear amplification product is obtained, and the 3'terminal nucleotide of the specific primer contains a bicolor functional group; linker connection is conducted on the linear amplification product, a linker connection product is obtained, and a linker connection system comprises single-chain ligase and a single-chain linker. The invention also discloses a kit / reagent for high-sensitivity detection of rare DNA molecular mutation. According to the method, rare mutant molecules with extremely low abundance in a sample can be detected with high sensitivity, and circulating tumor DNA molecules in a blood sample of an early-stage cancer patient can be detected at the same time.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to the application of single-primer amplification and library building technology in detecting the mutation of fragmented rare DNA molecules, and a reagent / kit. Background technique [0002] DNA mutation is the main cause of many diseases, and cancer is one of the diseases caused by DNA mutation. Through mutation detection, cancer patients can be molecularly typed, so as to treat symptomatically and obtain better curative effect. Next-generation sequencing (NGS) is a commonly used mutation detection technique. In the NGS detection process, it is generally necessary to amplify or enrich the target DNA to ensure that the number and concentration of target DNA molecules can be detected by the NGS sequencer; at the same time, it is also necessary to add sequencing on both ends of the target DNA molecules The sequence can be clustered and amplified on the sequencing carrier by the sequencing...

AI Technical Summary

Problems solved by technology

For the mutations of rare DNA molecules, PCR-based sequencing cannot be effectively detected, and false positive / false negative results are likely to be formed. The reasons are as follows: 1. The characteristics of PCR exponential amplification (the product generated by each round of amplification can become the next rounds of amplification templates) are prone to accumulating introduced error mutations, affecting the detection accuracy

2. In terms of steps, adapter ligation is generally performed first, and then PCR amplification is performed, because the efficiency of the ligase is limited and it is easy to cause missed detection

3. The sensitivity of PCR detection is limited by the ratio of the length of the nucleic acid molecule to be tested to the length of the PCR amplicon. In actual detection, considering the difficulty of designing primers and detection probes, usually for a DNA molecule of about 100-200bp, its The actual detection efficiency is about 20%, far below the level of a single molecule

5. Generally, single-end sample tags are used, which is easily affected by the production quality of sample tags and reduces the detection accuracy of rare DNA molecular mutations (limited by the production process, sample tags have about 1 / 1,000 base errors, which can easily lead to Analytical issues with sequencing data, affecting the detection of rare DNA molecules)

6. Generally, the molecular tag sequence is not used for deduplication analysis, and the wrong mutations introduced during library construction / sequencing cannot be removed

3) Off-target amplification caused by high-fidelity polymerase. Generally, as long as there are mismatched bases at the 3' end of the DNA sequence bound by the primer off-target, the amplification efficiency will be significantly reduced.

[0006] Limited by the above problems, existing technologies cannot effectively detect rare mutations

Therefore, the application of NGS is generally limited to the companion diagnosis of advanced cancer patients, and cannot effectively detect rare mutant DNA molecules in the blood of early and mid-stage cancer patients.

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