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Method for extending DNA sequence by cyclical crosses

A DNA sequencing and DNA sequence technology, applied in the biological field, can solve the problems of increasing the information of erroneous extension, accumulating the amount of markers, and reducing the number of templates, so as to increase the information of erroneous extension, the determination of the sequence is accurate, and the cumulative effect is not serious. Effect

Inactive Publication Date: 2009-10-21
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 extending DNA sequence by cyclical crosses
  • Method for extending DNA sequence by cyclical crosses

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] Refer to attached figure 1 , Design a pair of PCR primers, one of which is modified with acrylamide group. PCR primers are used to amplify a DNA sequence fragment containing SNP site number rs 11053646 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 polymerization is...

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] Refer to attached figure 1 , prepare the sequencing template according to the method of Example 1, and complete the 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 den...

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Abstract

The cyclic hybridization extension DNA sequencing method is a method that realizes the removal of extended markers, and involves a cyclic hybridization-extension DNA sequencing method. The steps are: Step 1) After the sequencing primer is hybridized with the fixed unknown DNA template, the On the sequencing primer, extend a labeled monomer at a time, and detect the base information in the extension of the unknown DNA template that determines different positions later; step 2) denature and separate the sequencing primer that extends the labeled monomer from the DNA template, and Re-hybridize the sequencing primers with the DNA template, use unlabeled monomers to extend to the determined base sequence, and then use labeled monomers to extend to continue sequencing. This method realizes the removal of extended markers, and each hybridization Sequencing primers are only extended to determine a small sequence, and the effect of wrong extension is not serious. The extension after re-hybridization is carried out according to the popular molecular biology method, which can always maintain the amount of DNA template and sequencing primer. The extension after re-hybridization is according to the popular molecular Biological methods, without the cumulative effect of the amount of markers, can always maintain the amount of DNA template and sequencing primers.

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 Patents(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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