Decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA)

Abstract

A decoding and sequencing method by real-time synthesis of two nucleotides into DNA. Single-sequencing reactions are performed on two different nucleotides, namely, X and Y simultaneously, and a base sequence fragment code XYn is obtained according to the quantitative relation between the number of synthesizing nucleotides and the number of the detected molecules which are generated in real time. The entire sequencing comprises performing two sets of sequencing reactions on the same template, wherein each set of sequencing comprises four nucleotides, namely, dATP, dCTP, dGTP, and dTTP, and cycles of sequencing reactions by simultaneous synthesis of two different nucleotides are performed in a manner that each nucleotide is used only once in each cycle. After several sequencing reactions, a set of XYn information ranked in a sequencing order is obtained. When said set of sequencing reactions is completed, denaturation is performed to eliminate an extended strand of a sequencing primer and the sequencing primer is re-crossed to perform a second set of sequencing reactions, and then several XYn information ranked by the second sequencing reaction is obtained; and finally, a specific base sequence of a nucleic acid fragment to be detected is assembled by decoding two sets of XYn information ranked in a sequencing order.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and is a method for realizing high-throughput determination of nucleic acid sequences, in particular to a two-nucleotide real-time synthetic DNA sequencing method and its application. Background technique

[0002] 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. Drastically reducing the cost of DNA sequencing will greatly promote research in life sciences and medicine, and even bring about revolutionary changes. At present, whole-genome DNA se...

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