DNA sequencing method using nucleotide and nucleotide with reversibly blocked end 3'

A DNA sequencing and nucleotide technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as unknown, achieve the effects of improving accuracy, improving sequencing accuracy, and reducing sequencing costs

Active Publication Date: 2018-06-15
SOUTHEAST UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0006] When only one nucleotide synthesis occurs in this sequencing reaction, it can be a synthesis reaction of X or a synthesis reaction of Y*, but i

Method used

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  • DNA sequencing method using nucleotide and nucleotide with reversibly blocked end 3'
  • DNA sequencing method using nucleotide and nucleotide with reversibly blocked end 3'
  • DNA sequencing method using nucleotide and nucleotide with reversibly blocked end 3'

Examples

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Embodiment 1

[0032] Example 1: A DNA sequencing method comprising nucleotides and 3' end reversibly blocked nucleotides was used to determine the artificially synthesized sequence comprising 3'-TAATCAG GTCTG-5' fragments.

[0033] 1. Template preparation: the artificially synthesized templates modified with 5' biotin were immobilized with avidin-modified magnetic beads, and then the magnetic beads were separated from the liquid, and the artificially synthesized templates immobilized on the magnetic beads were used for hybridization with sequencing primers.

[0034] 2. Hybridization of sequencing primers: Incubate the designed sequencing primers with the template immobilized by magnetic beads at 75°C for 5 minutes, then cool naturally to room temperature, then separate the magnetic beads from the liquid, and use the template immobilized by magnetic beads for DNA sequencing.

[0035] 3. Place the template immobilized by magnetic beads in the reactor (block both ends of the reactor with a semi...

Embodiment 2

[0058] Example 2: Two-nucleotide real-time synthetic DNA decoding and sequencing of the Escherichia coli genome

[0059] 1. Preparation of the whole genome template: the Escherichia coli genome is broken into fragments with a size of 100-1000 bp bases by ultrasound, and these fragmented nucleic acid sequences are connected with a pair of universal linkers with known sequences (such as : The sequence of the linker 1 is: CTG CTG TAC CGT ACA GCC TTG GCC G; the sequence of the linker 2 is: CGC TTT CCTCTC TAT GGG CAG TCG GTGA T) for ligation and pre-amplification for 10 cycles; then gel The 200-800bp DNA fragment was cut by electrophoresis and purified. These 200-800bp DNA fragments were subjected to emulsion parallel PCR reaction with microbeads immobilized with complementary sequences of one of the linkers to amplify the fragmented Escherichia coli genome fragments and denature them to obtain Escherichia coli genome sequencing DNA templates Beads of double-stranded DNA templates...

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Abstract

The invention discloses a DNA sequencing method using a nucleotide and a nucleotide with a reversibly blocked end 3'. X and Y* nucleotides simultaneously undergo a single sequencing reaction, whereinX represents a nucleotide with an unblocked end 3' and Y* is a nucleotide with a reversibly blocked end 3'. According to a quantitative relationship between the same number of detected molecules produced by two different nucleotides in the sequencing reaction and the number N of synthetic nucleotides, the sequencing information for a single sequencing reaction includes (N-1) specific bases X and 1or 0 coding XY. The whole sequencing process includes at least two sequencing reactions on the same template. Through comparing the two sets of sequencing information, the specific base sequence of the nucleic acid fragment to be tested is determined. Three groups of sequencing can also be performed. Through comparing the sequencing information obtained by the three sequencing reactions, the specific base sequences of the nucleic acid fragments to be tested are determined and the accuracy of sequencing is further improved.

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 DNA sequencing method comprising nucleotides and 3' end reversibly blocked nucleotides 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. A...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869
Inventor 肖鹏峰陈默然王明琛龚音简柏樑
Owner SOUTHEAST UNIV
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