A method for primer selection, sequencing by synthesis, and analysis of haplotypes of PCR products

A technology of synthetic sequencing and haplotype, which is applied in the fields of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve problems such as low efficiency, different amplification efficiency, and optimization design limitations of amplification primers

A technology of synthetic sequencing and haplotype, which is applied in the fields of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve problems such as low efficiency, different amplification efficiency, and optimization design limitations of amplification primers

CN104313168BActive Publication Date: 2016-11-30SOUTHEAST UNIV
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  • A method for primer selection, sequencing by synthesis, and analysis of haplotypes of PCR products
  • A method for primer selection, sequencing by synthesis, and analysis of haplotypes of PCR products
  • A method for primer selection, sequencing by synthesis, and analysis of haplotypes of PCR products

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example 1

[0032] Example 1: MMP7 (U25346) gene PCR product comprises the haplotype analysis of A / G (rs11568818) and C / T (rs11568819) nucleic acid fragments

[0033] (1) Genomic DNA in peripheral blood was extracted from the blood sample using traditional protein kinase K and phenol / chloroform extraction methods;

[0034] (2) PCR primers (5'-AGTCTACAGAACTTTGAAAGTATGTG-3', 5'-biotin-CTATGAGAGCAGTCATTTGACTTTG-3', 200 ng genomic DNA, 0.2mM dNTP, 1 U Taq DNA polymerase, 1×amplification buffer, 1.8 mM MgCl 2 50 μL PCR amplification system for amplification. The amplification conditions are: initial denaturation at 94°C for 5 minutes, 35 thermal cycles: denaturation at 94°C for 30 seconds, annealing at 61°C for 45 seconds, extension at 72°C for 45 seconds, and finally extension at 72°C for 7 minutes.

[0035] (3) React the PCR amplification with avidin-modified magnetic beads, immobilize the biotin-modified DNA strand on the magnetic beads, denature it under 0.2M NaOH solution, remove the un...

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Abstract

The invention provides a method for analyzing haplotype of PCR products by primer selection sequencing-by-synthesis. The method is adopted to perform haplotype analysis on two or more PCR products (multiple DNA templates) of site variation (such as SNP and mutation), wherein the PCR products are hybridized by a specific sequencing primer and then are divided into two parts, and a 3' terminus-closed monomer reagent is added to each part; the sequencing primer extends one base with the varied site under the guidance of a DNA template, the 3' terminus of the sequencing primer is closed and cannot extend continuously, and then the sequencing-by-synthesis is performed on the unclosed primers to obtain the sequence information of a specific DNA template, thereby determining the haplotype of PCR products.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for analyzing PCR product haplotypes by using primers to select, synthesize and sequence. Background technique [0002] In molecular biology research, DNA sequence analysis is the basis for further research and modification of target genes, and is crucial to life science. With the development and completion of the Human Genome Project and various model organism genome projects, human beings have entered the post-gene era, and the related disciplines of molecular biology have developed rapidly. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. In terms of basic research, research on the inheritance rules of disease genes and clone disease-causing genes; in terms of application, directly find the mutation sites of susceptibility genes of dis...

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Application Information

Patent Timeline
30 Nov 2016
Publication
CN104313168B
IPC
C12Q1/68
CPC
C12Q1/6869; C12Q2535/101; C12Q2565/301
Inventors
肖鹏峰; 潘荣芳