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A dna sequencing method for shortening dna template and its application

A DNA sequencing and template technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of poor sequencing accuracy and achieve high accuracy, high sequencing accuracy, and improved read length.

Inactive Publication Date: 2014-10-08
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, regardless of the synthesis or ligation sequencing method, the efficiency cannot always reach 100%. Each step of the sequencing reaction will consume a certain amount of "correct" sequencing primers and produce some "wrong" sequencing primers. Therefore, as the synthesis (or As the number of connections) increases, the accumulated errors will increase, and the accuracy of sequencing is given by the correctly synthesized (or connected) marker signal, so the accuracy of sequencing is getting worse

Method used

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  • A dna sequencing method for shortening dna template and its application
  • A dna sequencing method for shortening dna template and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Determination of human genome by ligation sequencing of shortened DNA template

[0057] (1) After the human genome was treated with Fok I enzyme at 37°C for 2 hours, it was ultrasonically broken into fragments with a size of 50-1000 bases, and these fragmented nucleic acid sequences were connected with a pair of universal linkers under the action of ligase Ligation (assumed to be 20 bases), wherein 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 contains the Mly I enzyme recognition sequence, and This specific region is located at the very beginning of template sequencing.

[0058] (2) The fragmented nucleic acid sequences connected by these tethers and the complementary sequences of the immobilized linkers are placed on microbeads to perform emulsion parallel PCR reaction to amplify the fragmented whole human genome. And these m...

Embodiment 2

[0071] Example 2: Determination of Escherichia coli genome by extension sequencing method of shortened DNA template

[0072] (1) After the E. coli genome was treated with Mme I enzyme at 37°C for 2 hours, it was ultrasonically broken into fragments with a size of 50-1000 bases, and these fragmented nucleic acid sequences were connected with a pair of universal ligation under the action of ligase The oligonucleotide sequence of one of the universal linkers is completely complementary to the sequence of the amplification primer, and the oligonucleotide sequence of the other linker contains the recognition sequence of the Mme I enzyme. And the specific region is located at the initial position of the template sequencing.

[0073] (2) The fragmented nucleic acid sequences connected by these tethers and the complementary sequences of the immobilized linkers are placed on microbeads to carry out emulsion parallel PCR reaction to amplify the fragmented Escherichia coli genome. And t...

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Abstract

The invention relates to a method for shortening DNA sequencing of a DNA template and application thereof. The method comprises the following steps of: (1) determining a small segment of sequence which is fixed in the DNA template by synthesizing or adopting a connecting sequencing method; (2) stopping the sequencing of sequencing primers, adding four kinds of dNTPs monomer extension sequencing primer strands, and making the DNA template become double strands; (3) shortening the DNA template: processing by using restriction enzyme to preset a connexon with an enzyme recognition sequence; and fracturing a small segment of sequence in the determined DNA template; (4) connecting the sequencing primer and a double stranded oligonucleotide segment with the enzyme recognition sequence on the cut and shortened DNA template; (5) denaturing, removing unfixed DNA strands, and obtaining the DNA template again; (6) hybridizing the sequencing primer, and continuously determining the small segment of sequence fixed in the DNA template; and (7) repeating the step (2) to the step (6) until the sequencing of the undermined DNA template is completed.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing in biotechnology, in particular to a DNA sequencing method for shortening a DNA template and its application, and is especially suitable for high-throughput DNA sequencing. technical background [0002] The development and completion of the Human Genome Project and various model organism genome projects has brought mankind into the post-gene era, which has had a huge impact on contemporary biological and medical research, and the related disciplines of molecular biology have developed rapidly. 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 shifted from whole-genome sequence determination to the comparison of individual genetic differences and inter-species genetic diffe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/19
Inventor 肖鹏峰浦丹谢宏梅王文捷陆祖宏
Owner SOUTHEAST UNIV
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