Application of oligonucleotide probe containing dithio nucleotide on detecting DNA sequences

The technology of dithionucleoside oligonucleotide and dithionucleoside is applied to DNA sequencing probe and its application field in determining DNA sequencing sequence, and can solve the problem of non-degrading single-stranded DNA double-stranded DNA performance, It can not cut the phosphodiester bond, does not degrade the phosphodiester bond, etc., to achieve the effect of accurate and reliable measurement, increased reading length, and accurate cutting position

Inactive Publication Date: 2009-10-07
SOUTHEAST UNIV
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Problems solved by technology

Exonuclease III can catalyze the reaction of removing single nucleotides one by one from the 3'-terminal hydroxyl of double-stranded DNA, but it does not degrade the phosphodiester bond in the nucleic acid, nor degrades single-stranded DNA and double-stranded DNA with 3'-end overhang performance
At the same time, existing studies have shown (Yang et al. Nucleic Acids Research, 2007, 35(9), 3118-3127), that the degradation of exonuclease III is hindered by thio-modified nucleosides, that is, they cannot phosphodiester bond cleavage

Method used

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  • Application of oligonucleotide probe containing dithio nucleotide on detecting DNA sequences
  • Application of oligonucleotide probe containing dithio nucleotide on detecting DNA sequences
  • Application of oligonucleotide probe containing dithio nucleotide on detecting DNA sequences

Examples

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Embodiment 1

[0053] Hybridization-ligation fluorescent-labeled sequencing assays encompass the entire human genome.

[0054] Cut the human genome with enzymes (or sonicate) into fragments with a size of 50-1000 bases, and use a pair of universal linkers (assumed to be 20 bases) to these fragmented nucleic acid sequences under the action of ligase T4 ligase (Shanghai Bioengineering Co., Ltd.) was used for ligation at 37° C. for 2 hours. The complementary oligonucleotide sequence of the linker is used as the amplification primer (one of which is pre-immobilized on the microbeads: the immobilization method can be a biotin-modified oligonucleotide synthesized by Yingjun (Shanghai) Bioengineering Co., Ltd. and New England Biolabs (USA) Co., Ltd. The produced streptavidin-modified microbeads were reacted at room temperature for 1 hour), and the sequence of one of the linker oligonucleotides was exactly the same as that of the sequencing primer in the subsequent sequencing reaction

[0055] The ...

example 2

[0072] Sequencing of three known DNA template sequences

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Abstract

The invention aims at providing an application of oligonucleotide probe containing dithio nucleotide on detecting DNA sequences, namely a nucleotide sequence detecting probe (namely containing dithio) without an enantiomorph structure which uses the degradation of excision enzyme II on the phosphodiester bonds between normal basic groups and the function of not being cut of the excision enzyme on the phosphodiester bonds modified by dithio to remove markers in the sequence detecting reaction and build a new hybridization-enzyme connection-enzyme cutting high flux sequence detecting technology. The nucleotide sequence detecting probe provides a new method for analyzing a genome DNA sequence, builds a quick, accurate and cheap genome sequence detecting technology, and has the advantages of accurate and reliable detecting and easy realization.

Description

1. Technical field [0001] The invention relates to a DNA sequencing probe and its application in determining the DNA sequence, in particular to a dithionucleoside-containing oligonucleotide sequencing probe and its application in determining the DNA sequence. 2. Background technology [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 ...

Claims

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Application Information

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