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Pre-capture primers, capture probes and construction methods, sequencing methods and applications for constructing human mitochondrial dna libraries

A DNA library and construction method technology, applied in the field of pre-capture primers, can solve the problems of long operation process, low detection efficiency, and complicated steps, and achieve the effect of reducing interference, reducing operation, and rapid capture

Active Publication Date: 2020-06-09
HANGZHOU JILUO BIOLOGICAL PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0010] The first object of the present invention is to provide pre-capture primers for constructing human mitochondrial DNA libraries, the second object of the present invention is to provide capture probes for constructing human mitochondrial DNA libraries, and the third object of the present invention is to To provide a method for constructing a human mitochondrial DNA library, the fourth object of the present invention is to provide a sequencing method for a human mitochondrial DNA library, and the fifth object of the present invention is to provide the above method in the analysis of human mitochondrial DNA heterogeneity In order to alleviate the technical problems of complicated steps, long operation process, strict instrument requirements and low detection efficiency in the prior art

Method used

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  • Pre-capture primers, capture probes and construction methods, sequencing methods and applications for constructing human mitochondrial dna libraries
  • Pre-capture primers, capture probes and construction methods, sequencing methods and applications for constructing human mitochondrial dna libraries
  • Pre-capture primers, capture probes and construction methods, sequencing methods and applications for constructing human mitochondrial dna libraries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Designs primers and probes

[0050] According to the needs of the invention, design pre-capture primers as shown in Table 1:

[0051] Table 1 Pre-capture primers

[0052] name sequence serial number E1Fa GGGAGCTCTCCATGCATTTG SEQ ID NO.1 E2Fa GCACACCCGTCTATGTAGCA SEQ ID NO.3 E3F TCCTACTCCTCCATTGTACCCA SEQ ID NO.5 E4F CCATCCTTACCCACCCTCGTT SEQ ID NO.7 E5F GAAAACCCCACAAACCCCATT SEQ ID NO.9 E6F AAGGACTGCAAAAACCCACT SEQ ID NO.11 E1Ra CAGGCGGTGCCTCTAATACT SEQ ID NO.2 E2R CCTGCGGCGTATTCGATGTT SEQ ID NO.4 E3R GTGGGTTTAAGTCCCATTGGT SEQ ID NO.6 E4R TGGTCGTGGTTGTAGTCCGT SEQ ID NO.8 E5Ra GGATGAGGCAGGAATCAAAGAC SEQ ID NO.10 E6R AAGGTGGATGCGACAATGGA SEQ ID NO.12

[0053] According to the needs of the invention, the design of capture probes and grouping is as follows:

[0054]Probe set 1: E1F has the sequence shown in SEQ ID NO.13, E1R has the seq...

Embodiment 2

[0055] Example 2 sample preparation DNA extraction

[0056] 1. Collection of specimens: 2 mL of peripheral venous blood of family members was collected after the approval of the ethics committee and the signing of the informed consent by the family members for genetic testing.

[0057] 2. Extraction of whole genome DNA: operate according to the operating instructions of the blood genome DNA extraction kit, the kit is DNeasyBlood&TissueKit.

Embodiment 3

[0058] Example 3 Construction of Human Mitochondrial DNA Library

[0059] (1) Pre-capture

[0060] Using the DNA extracted in Example 2 as a template, the 6 pairs of pre-capture primers (shown in Table 1) provided in Example 1 of the present invention were respectively used for capture PCR to obtain target sequence pre-capture products. See Table 3 for the pre-capture reaction system.

[0061] Table 3 Pre-capture system

[0062] Reagent volume 10×PCR Buffer with Mg 2+

2.5μL dNTP Mixture (2.5mM each) 2μL Taq DNA polymerase (5U / μL) 0.125μL Upstream primer (10μM) 1μL Downstream primer (10μM) 1μL template DNA 2μL wxya 2 o

16.375μL

[0063] The reaction conditions were 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 5 min, and a total of 30 cycles of amplification; finally, extension at 72°C for 10 min. The result is as figure 2 with image 3 shown, from ...

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Abstract

The invention provides a pre-capture primer and a capture probe for building a human mitochondrial DNA library as well as a building method, a sequencing method and application, and relates to the technical field of genomics. The pre-capture primer and the capture probe for building the human mitochondrial DNA library provided by the invention comprise sequences shown in a sequence table, so that quick capture on a human mitochondrial sequence can be realized. With the building method provided by the invention, the interference of homologous sequences in a genome DNA on mitochondrial sequence analysis can be reduced; when the library is built, a three-step PCR (polymerase chain reaction) method is only carried out, processes including screening of a target sequence, capture of the targeted sequence and introduction of adaptor sequence can be completed, thereby reducing the operation difficulty and improving the experimental efficiency; moreover, because main processes of the building method disclosed by the invention are all completed on a PCR apparatus, a reagent is convenient to use and the cost is low, the implementation of the building method can be completed in a common laboratory.

Description

technical field [0001] The invention relates to the technical field of genomics, in particular to a pre-capture primer, a capture probe, a construction method, a sequencing method and an application for constructing a human mitochondrial DNA library. Background technique [0002] Mitochondria are very important organelles in eukaryotic cells, the main part of eukaryotic oxidative metabolism, and the place where carbohydrates and other substances are finally oxidized in cells to release energy. Mitochondria itself has an independent set of genome, which only divides in oocytes, so it has the characteristics of maternal inheritance, that is, it is only passed on to the next generation through women. The transmission of mtDNA mutations has a number of features. [0003] Each cell contains a large number of mitochondria. If a certain site on all the mitochondrial DNA in the cell is the same type of base, that is, all are normal genes or mutant genes, the cell is pure; if A cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C40B50/06C12Q1/6806C12Q1/6869
CPCC12N15/1093C12N15/11C12Q1/6806C12Q1/6869C40B50/06C12Q2525/191C12Q2531/113
Inventor 范嘉庚王伟李洲
Owner HANGZHOU JILUO BIOLOGICAL PHARMA CO LTD
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