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Sequencing method for reverse hybridized coupling extended DNA

A DNA sequencing and DNA sequence technology, applied in the field of DNA sequencing, can solve the problems of false positives and high cost, large amount of probe synthesis, and high false positives, and achieves solutions to false positives and high cost, reliable accuracy and low cost. Effect

Inactive Publication Date: 2010-01-20
SOUTHEAST UNIV
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] Technical problem: The purpose of the present invention is to provide a reverse hybridization coupling extension DNA sequencing method, which can overcome the defects of high false positives in the traditional hybridization sequencing method, and solve the problem of large amount of probe synthesis and high cost in the existing method, Achieve high-throughput, low-cost, high-sensitivity and rapid determination of short DNA sequences
That is, it is realized by repeating the steps of "hybridization-extension-detection" on the DNA template, that is, adding an extension step to the traditional hybridization sequencing method, which can solve the problems of false positives and high cost

Method used

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  • Sequencing method for reverse hybridized coupling extended DNA
  • Sequencing method for reverse hybridized coupling extended DNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A DNA sequence to be tested is amplified by conventional PCR and fixed on an acrylamide substrate to make an unknown DNA template. Prepare 64 hybrid primers, with three known bases at the 3' end and 8 bases at the 5' end. The sequence characteristics of the hybrid primers can be expressed as 5'NNNNNNNNNXYZ 3', where N is four bases T, A , any one of G, C not limited, or can be replaced by universal base I, X, Y, Z are all selected from T, A, G, a certain base in C, such as NNNNNNNNACG, NNNNNNNNGAG etc., XYZ constituted 64 different sequences in the hybridization primers.

[0036] 1) Hybridization: hybridize the hybridization primer NNNNNNNNACG with the immobilized unknown DNA template. Dissolve the hybridization primers at 100uM concentration in 1X hybridization solution, take an appropriate amount (submerge the template to be tested completely) and place the hybridization primers in the template to be tested, denature at 60°C for 2 minutes, then anneal at 4°C for 3 mi...

Embodiment 2

[0043] Whole-genome sequencing of a single human by reverse hybridization-coupled extension DNA sequencing

[0044] DNA templates can be prepared as follows:

[0045] 1. Extract the human genome.

[0046] 2. DNA fragmentation. Use ultrasonic treatment to make the average size of DNA around 50bp.

[0047] 3. Add linker primer a. Firstly, the fragmented genomic DNA is blunted with polymerase, and its 3-terminus is treated with a protruding A. Then, under the action of ligase, the linker primer with the Mme I endonuclease recognition site is connected to the small fragment of DNA.

[0048] 4. Enzyme digestion treatment. Under the action of Mme I endonuclease, cut the whole genome into a genomic library with a fragment size of 19 bp.

[0049] 5. Same as step 3, add adapter primer b to the other end.

[0050] 6. Microemulsion PCR amplifies a single-clonal genome library.

[0051] 7. Whole genome microarray fabrication. Fix the amplified product from the previous step on an...

Embodiment 3

[0054] RNA Expression Profiling by Reverse Hybridization Coupled with Extended DNA Sequencing

[0055] DNA templates can be prepared as follows:

[0056] 1. Extract mRNA from normal cells and diseased cells respectively, and use magnetic beads to fix 30 T nucleotide chains to purify mRNA so that the magnetic beads can absorb mRNA.

[0057] 2. Reverse transcribe into cDNA, and use RNaseH, DNA polymerase, etc. to synthesize the second strand.

[0058] 3. Digest the product of step 2 with an endonuclease (Sau 3AI) that recognizes four bases. Wash the magnetic beads with binding buffer.

[0059] 4. Add linker primer A (with endonuclease Acu I recognition site on the primer) under the action of ligase.

[0060] 5. Digest the product of step 4 with Acu I. A cDNA library of adapter primer A followed by 16 unknown sequences to be tested was obtained.

[0061] 6. Add another linker primer B. Amplification was then performed using emulsion PCR.

[0062] 7. The amplified product i...

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Abstract

The present invention discloses a reverse hybridization coupling elongation DNA sequencing method. Said method includes the following steps: making unknown DNA template be parallelly hybridized with several probes whose 3' tail end has three or more than three known bases and elongating labeled base, detecting that the label is elongated or not, utilizing base complementation principle to define that the unknown DNA template has correspondent information with four or more than four continuous bases or not, and splicing the DNA sequence information to be detected. Said invention can repeatedly make the steps of hybridization, elongation and detection, etc. and can be used for quickly determining short fragment DNA sequence.

Description

technical field [0001] The present invention relates to a DNA sequencing method, in particular to a DNA hybrid sequencing method. Background technique [0002] With the deepening of genome research, it will become possible to understand the differences in life, the rules of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Although numerous factors contribute to the occurrence of disease, genetic mutations (single nucleotide polymorphisms, methylation, etc.) are widely recognized as an important intrinsic factor. In terms of basic research, gene mutation sites are helpful for the precise positioning of genomes, the study of genetic laws of disease genes, and the cloning of disease-causing genes; in terms of applications, it is of great value to directly find disease-susceptible gene mutation sites. Most complex diseases, such as cancer, diabetes, cardiovascular disease, depression, asthma, etc., are affected by ...

Claims

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Application Information

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