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Method for extensional sequencing DNA by circular crossbreed

A technology of DNA sequencing and DNA sequence, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of increasing error extension information, accumulation of markers, reducing the number of templates, etc., to achieve the goal of increasing error extension Accurate determination of information and sequence, and the effect of not serious cumulative effect

Inactive Publication Date: 2007-07-25
SOUTHEAST UNIV
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Problems solved by technology

Since the connection between the marker and the monomer is a stable chemical bond, when the marker is destroyed or cut by chemical or light methods, it will undoubtedly cause damage to the DNA template and sequencing primers, reduce the number of templates and increase false extensions information; marker damage or incomplete cutting will lead to the accumulation of the amount of markers, making it difficult to determine the amount of subsequent markers, affecting the length and accuracy of sequencing

Method used

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  • Method for extensional sequencing DNA by circular crossbreed
  • Method for extensional sequencing DNA by circular crossbreed

Examples

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example 1

[0022] Example 1: The cycle hybridization-fluorescence extension sequencing method determines a DNA fragment containing the SNP site number rs11053646 in the human genome,

[0023] With reference to accompanying drawing 1, design a pair of PCR primer, wherein a primer has modified acrylamide group. PCR primers are used to amplify a DNA sequence fragment containing SNP site number rs11053646 in human samples (such as blood, saliva, etc.). The PCR product is fixed on a glass slide by polymerization method, and the unfixed PCR chain and other impurities are removed by denaturation and electrophoresis methods to obtain a pure DNA chain. One primer, unmodified, hybridizes to the immobilized DNA template.

[0024] Under the extension reaction conditions, according to the order of A, G, C, T, one base is added each time, and the fluorescently labeled monomer is added for one round, until the change in fluorescence is added each time after the labeled monomer is added and the polymer...

example 2

[0027] Example 2: Determination of a DNA fragment comprising the SNP site number rs11053646 in the human genome by cycle hybridization-pyrosequencing.

[0028] Referring to accompanying drawing 1, prepare the sequencing template according to the method of Example 1, and complete hybridization with the sequencing primer.

[0029] According to the order of A, G, C, T, add one base each time and add unlabeled monomers respectively, and judge whether the extension and the light intensity of the base occur according to whether the light and light intensity are generated in the pyrosequencing system under the extension reaction conditions The number of extensions, so it can be inferred that the DNA template sequence determined in this hybridization is AAG ACT GGA TCTGGC ATG GAG AAA ACT G. When the amount of light change cannot reflect the number of extended bases, the pyrosequencing of this hybridization is stopped.

[0030] The sequencing primers extending the above 28 bases were ...

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Abstract

This invention relates to a method for sequencing DNA through cycled hybridization-elongation. The method comprises: (1) hybridizing sequencing primers with the immobilized unknown DNA template, elongating the sequencing primers with a marked single alkali, and detecting the information of the added alkali; (2) denaturalizing and separating the elongated sequencing primers from the DNA template, hybridizing the DNA template with the sequencing primers over again, elongating to the defined alkali sequence with unmarked single alkali, and then elongating with marked single alkali. The method realizes the removal of elongation markers, and has little error elongation and no accumulation effect of markers amount.

Description

technical field [0001] The invention is a method for removing extended markers, relates to a cycle hybridization-extended DNA sequencing method, and belongs to the field of biotechnology. technical background [0002] With the deepening of genome research, it will become possible to understand the differences in life, the rules of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Although numerous factors contribute to the occurrence of disease, genetic mutations (single nucleotide polymorphisms, methylation, etc.) are widely recognized as an important intrinsic factor. In terms of basic research, gene mutation sites are helpful for the precise positioning of the genome, the study of the genetic law of disease genes, and the cloning of disease-causing genes; Cancer, diabetes, cardiovascular disease, depression, asthma, etc. are affected by many genes and environmental factors. Through large-scale identification ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 肖鹏峰陆祖宏白云飞吕华孙啸谢建明
Owner SOUTHEAST UNIV
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