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Primer, method and kit for constructing gene mutation sequencing library

A sequencing library and gene technology, applied in the field of molecular biology, can solve problems such as mismatches

Active Publication Date: 2015-09-30
广州益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the parallel detection of multiple genes based on multiplex PCR fails to solve the following problems: secondary structures such as hairpin structures or dimers are formed inside the primers; there is non-specific binding between the designed primers, and the combination of DNA tags forms Dimers formed between index primers, mismatches, non-specific binding to PCR products, etc.

Method used

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  • Primer, method and kit for constructing gene mutation sequencing library
  • Primer, method and kit for constructing gene mutation sequencing library
  • Primer, method and kit for constructing gene mutation sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of primers for gene mutation sequencing library

[0042] 1. Amplify the primers with the target sequence containing the gene mutation site

[0043] The present invention provides amplification primers for 47 target genes, and the corresponding amplification primers can be selected according to the types of detected genes and mutation sites, as shown in Table 1.

[0044] Table 1 Amplification primers

[0045]

[0046]

[0047]

[0048]

[0049]

Embodiment 2

[0050] Example 2 DNA tag sequence and tag primer

[0051]The DNA tag used in the present invention refers to a short base sequence added at the 5' end of the PCR primer, for the PCR primers of each sample, at least one of each pair of primers is connected to a specific DNA tag , so as to form DNA tag primers, and through PCR amplification, the PCR products from the same sample source can be equipped with corresponding DNA tags, thereby accurately characterizing the sample source of DNA through DNA tags, and by combining multiple PCR products from different sample sources ( with different DNA tags) are mixed into a library, thus realizing high-throughput sequencing of multiple samples in one sequencing.

[0052] Each pair of index primers consists of two parts, at least one of the two primers is a DNA tag at the 5' end and an amplification primer at the 3' end, wherein the DNA tag is used to mark the PCR product in the PCR amplification reaction, and the amplification primer P...

Embodiment 3

[0059] Example 3 DNA labeling kit for constructing sequencing library

[0060] The DNA tag construction sequencing library kit of the present invention includes: different sets of PCR primers for different samples, each set of PCR primers includes at least one pair of PCR primers, and the 5' of at least one of each pair of PCR primers DNA tag sequences are connected at the ends, the DNA tag sequences of the same primer set are the same, and the DNA tag sequences of different primer sets are different, the DNA tag sequences are selected from Example 2, and the PCR primers are selected from Example 1.

[0061] In this embodiment, the amplification primers (Table 1) in Example 1 and the DNA tags (Table 2) in Example 2 are used to construct tagged PCR primers. In this embodiment, there are 20 kits for constructing sequencing libraries with DNA tags. Group label PCR primer set, each group label PCR primer all comprises all primers of table 1 in embodiment 1, and the 5' end of forwa...

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Abstract

The invention relates to a PCR primer involving 47 genes and a tag PCR primer. The tag PCR primer is composed of the PCR primer and a DNA tag sequence connected to the 5' end of at least one primer in the PCR primer pair, wherein the DNA tag sequence is selected from SEQ ID NO: 107 to SEQ ID NO: 126. The invention further relates to a method and a kit for constructing a sequencing library by applying the DNA tag of the tag PCR primer. The coincidence rates of the detection method provided by the invention and the sequencing method is up to 100%, the provided amplification primer and the DNA tag form a detection product with a specificity, a sensitivity and a repeatability which are optimized and balanced, somatic mutation and mononucleotide polymorphism can be detected in parallel, and a plurality of base sequences of 1-20 sample sources can be accurately differentiated by one-time detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a primer, a method and a kit for constructing a gene mutation sequencing library. technical background [0002] Gene mutation refers to the change of base pair composition or sequence in gene structure. Common gene mutations include somatic mutation and single nucleotide polymorphism (single nucleotide polymorphism, SNP). [0003] Somatic mutations are mutations that occur in normal body cells, such as in the skin or organs. In recent years, studies on cancer genomes have found that there are tens of thousands of somatic mutation sites related to the process of carcinogenesis. However, current cancer diagnosis and treatment often ignore this point. Clinical treatment strategies are mainly aimed at the mutant cells with a dominant number. Take the monoclonal antibody drug Herceptin for the treatment of breast cancer as an exam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/68
Inventor 刘志明吴诗扬廖传荣
Owner 广州益善医学检验所有限公司
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