Method for improving specificity in cutting position of endonuclease V

An endonuclease and specific technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as error and sequencing speed limit, achieve high cutting efficiency, increase read length, and cut position accurate effect

Inactive Publication Date: 2010-04-14
SOUTHEAST UNIV
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Problems solved by technology

Errors in the position of the sequencing bases will be introduced during the sequencing process, which will greatly limit the accuracy of the sequencing

Method used

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  • Method for improving specificity in cutting position of endonuclease V
  • Method for improving specificity in cutting position of endonuclease V
  • Method for improving specificity in cutting position of endonuclease V

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example 1

[0033] Example 1: Hybridization-ligation Fluorescently Labeled Sequencing Determination of Inclusion of the Whole Human Genome.

[0034] Cut the human genome with enzymes (or sonicate) into fragments with a size of 50-1000 bases, and connect these fragmented nucleic acid sequences with a pair of universal linkers (assumed to be 20 bases in length) under the action of ligase Base), the oligonucleotide sequence of one universal linker is completely complementary to the sequence of the amplification primer, and the oligonucleotide sequence of the other linker is the same as that of the sequencing positioning primer.

[0035] The fragmented nucleic acid sequence connected by these tethers and the complementary sequence of the fixed linker are transferred to microbeads for emulsion parallel PCR reaction to amplify the fragmented whole human genome. These microbeads are immobilized on the plate substrate, and the human whole genome sequencing template is obtained by enzyme digestion...

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Abstract

The method provides a method for improving the specificity in a cutting position of endonuclease V and relates to a high-throughput method for analyzing DNA sequences, in particular to a high-speed low-cost and high-throughput method for DNA sequencing. Recognition-site deoxyinosine dI containing the endonuclease V and DNA segments of single-chain oligonucleotide at a site where oxygen on one or more phosphodiester bonds in a DNA chain is sulfo-modified are introduced into a DNA probe, and the distance between the recognition site of the endonuclease V and the sulfo-modified site is two or more basic groups. The method can be used for analyzing unknown DNA, namely sequence information of a template DNA to be analyzed. Re-hybridized high-throughput primers are obtained by synthesis and purification by a very mature solid-phase DNA method, and therefore, the method does not have wrongly extended accumulative effect and can maintain the amount of DNA templates and sequencing primers. The determination of the sequence is correct and reliable, having no limitations on sequenced length.

Description

technical field [0001] The invention relates to a high-throughput DNA sequence analysis method, in particular to a fast, low-cost and high-throughput DNA sequence method. Belongs to the field of biotechnology. Background technique [0002] Nucleic acid (such as DNA) contains the blueprint of all living organisms, and the study of nucleic acid sequences is crucial to life sciences. With the development and completion of the Human Genome Project and various model organism genome projects, human beings have entered the post-gene era, which has had a huge impact on contemporary biological research and medical research, and the related disciplines of molecular biology have been rapidly developed. develop. 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 shift...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陆祖宏李燕强肖鹏峰
Owner SOUTHEAST UNIV
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