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Two-nucleotide synthetic sequencing analysis method for multi-template PCR product

A two-nucleotide, sequencing-by-synthesis technology, used in biochemical equipment and methods, and microbial determination/inspection. Effect

Active Publication Date: 2015-09-09
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the invention patent only analyzes one DNA template sample, and cannot analyze sequences containing multiple template DNAs, which limits the scope of sequence analysis

Method used

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  • Two-nucleotide synthetic sequencing analysis method for multi-template PCR product
  • Two-nucleotide synthetic sequencing analysis method for multi-template PCR product
  • Two-nucleotide synthetic sequencing analysis method for multi-template PCR product

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Experimental program
Comparison scheme
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Embodiment 1

[0047] Embodiment 1: The human sample comprises the PCR product DNA template analysis method of MMP7 (U25346) gene comprising a SNP (C / T, rs11568819 site, and specific method comprises:

[0048] (1) Genomic DNA in peripheral blood was extracted from the blood sample using traditional protein kinase K and phenol / chloroform extraction methods;

[0049] (2) Combine PCR primers 5'- agtctacaga actttgaaag tatgtg -3', 5'-biotin- ctatgagagc agtcatttga ctttg -3' with 200 mg genomic DNA, 0.2 mM dNTP, 1 U Taq DNA polymerase, 1× amplification buffer , 1.8 mM MgCl 2 The 50 μL PCR amplification system was used for amplification. The amplification conditions were: initial denaturation at 94 °C for 5 min; 35 thermal cycles: denaturation at 94 °C for 30 s, annealing at 61 °C for 45 s, and extension at 72 °C for 45 s; Extend at 72 °C for 7 min;

[0050] (3) Divide the PCR amplification product evenly into two parts, and carry out the operations of steps (4) to (6) respectively;

[0051] (4) ...

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Abstract

The invention discloses a two-nucleotide synthetic sequencing analysis method for a multi-template PCR product. The method comprises the steps of uniformly dividing the same PCR product into at least two parts, and carrying out two-nucleotide synthetic sequencing, wherein each sequencing reaction obtains sequencing encoding information containing two nucleotide kinds and nucleotide synthetic amount; selecting at least two groups of encoding information respectively obtained by at least two groups of different two-nucleotide synthetic circular sequencing in three groups to decode; and finally, determining the contents of different DNA templates of the PCR product through association analysis. The two-nucleotide synthetic sequencing analysis method can be used for widening the single-nucleotide synthetic sequencing analysis range, directly measuring the proportions of all the DNA templates in a sample and finding and screening nucleotide markers from large-scale samples, and is suitable for sequencing analysis of the multi-template PCR product and a mixed sample PCR product.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a PCR product sequence analysis method, in particular to a method for analyzing multi-template PCR products by two nucleotide synthesis sequencing. Background technique [0002] The development and completion of the Human Genome Project and various model organism genome projects has brought mankind into the post-gene era, which has had a huge impact on contemporary biological and medical research, 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. As far as gene sequence analysis is concerned, the focus of the post-genome era has shifted from whole-genome sequence determination to the comparison of individual genetic differences and inter-species genetic differences in the genome....

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869
Inventor 肖鹏峰浦丹潘荣芳殷豪景
Owner SOUTHEAST UNIV
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