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Hybrid high-throughput DNA sequencing method based on ligase reaction

A DNA sequencing and ligase reaction technology, applied in the field of DNA sequencing, can solve the problems of large amount of probe synthesis, short hybridization probes, high false positives, etc., to solve false positives and high cost, low cost, and reliable accuracy Effect

Inactive Publication Date: 2017-06-16
XUZHOU MEDICAL COLLEGE
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Problems solved by technology

[0005] The invention provides a hybridization high-throughput DNA sequencing method based on ligase reaction. The purpose is to overcome the defects of high false positives in the traditional hybridization sequencing method, and to solve the problems of large amount of probe synthesis, high cost and short hybridization probes in the existing method. To achieve high-throughput, low-cost, high-sensitivity and rapid determination of short-segment DNA sequences

Method used

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  • Hybrid high-throughput DNA sequencing method based on ligase reaction
  • Hybrid high-throughput DNA sequencing method based on ligase reaction
  • Hybrid high-throughput DNA sequencing method based on ligase reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A DNA sequence to be tested is amplified by conventional PCR and fixed on an aldehyde substrate to make an unknown DNA template. Prepare 256 DNA hybridization primers with a length of 8 bases, with four known bases at the 3' end and 4 random bases at the 5' end. The sequence characteristics of the hybridization primers can be expressed as 5'NNNNWXYZ3', where N is Any one of the four bases T, A, G, and C is not limited, or it can be replaced by a universal base I, and W, X, Y, and Z are all selected from T, A, G, and C. One base, such as 5'NNNNACGG3', 5'NNNNGACG3', etc., has 256 different sequences composed of WXYZ in hybridization primers.

[0035]1) Hybridization: The hybridization primer 5'NNNNGACG3' is hybridized with a fixed, 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 ...

Embodiment 2

[0041] Example 2: Hybridization-Joint Fluorescent Labeled Sequencing Method Determination of Inclusion of the Whole Human Genome.

[0042] Cut the human genome with enzymes (or sonicate) into fragments with a size of 50-1000 bases, and connect these fragmented nucleic acid sequences with a pair of universal linkers (assumed to be 20 bases in length) under the action of ligase Base), 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 is the same as that of the sequencing positioning primer. The fragmented nucleic acid sequence connected by these tethers and the complementary sequence of the fixed linker are transferred to microbeads for emulsion parallel PCR reaction to amplify the fragmented whole human genome. These microbeads are immobilized on the plate substrate, and the human whole genome sequencing template is obtained by enzyme digestion or den...

Embodiment 3

[0044] Example 3: RNA expression profiling analysis based on ligase reaction-based hybrid high-throughput DNA sequencing method

[0045] 1. Extract the mRNA of normal cells and diseased cells respectively, and use magnetic beads to immobilize 30 T nucleotide chains to purify the mRNA so that the magnetic beads can absorb mRNA.

[0046] 2. Reverse transcribe into cDNA, and use RNaseH, DNA polymerase, etc. to synthesize the second strand.

[0047] 3. Digest the product of step 2 with an endonuclease (CSau 3AI) that recognizes four bases. Wash the magnetic beads with binding buffer.

[0048] 4. Add linker primer A (with endonuclease Acu I recognition site on the primer) under the action of ligase.

[0049] 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.

[0050] 6. Add another linker primer B. Amplification was then performed using emulsion PCR.

[0051] 7. The amplified product is im...

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Abstract

The invention belongs to the technical fields of biology and medicine, and relates to a hybridization high-flux DNA (Deoxyribose Nucleic Acid) sequencing method based on ligase reaction. The hybridization high-flux DNA sequencing method disclosed by the invention can overcome the defect of high false positive of the traditional hybridization sequencing method, solve the problems of large probe synthetic quantity, high cost, short hybridization probe and the like of the traditional method and realize the high-flux, the low cost and the high sensitivity and is capable of fast determining the DNA sequence of a short fragment.

Description

technical field [0001] The invention relates to a DNA sequencing method, in particular to a hybrid high-throughput DNA sequencing method based on ligase reaction. Background technique [0002] New DNA sequencing technologies currently under development are mainly focused on non-electrophoretic means. On the whole, such technologies can be divided into four categories: the first category is sequencing-by-synthesis, which detects during the process of adding bases to the extended DNA chain; the second category is hybrid sequencing, which prepares a set of high-density The hybridization signal of the oligonucleotide microarray chip is used to identify the sequence of the target gene; the third type is molecular imaging technology, a series of technologies that can be sequenced at the single-molecule level; the last type of technology is to induce DNA molecule winding Through very small holes, the bases are read out by means of electronic or optical methods in this process, als...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2533/107C12Q2521/501C12Q2535/122
Inventor 李燕强郭羽白潘志强曹君利
Owner XUZHOU MEDICAL COLLEGE
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