DNA sequencing method based on primer array chip

A DNA sequencing and array chip technology, applied in the field of DNA sequencing, can solve the problems of high cost, increase reliability, and high false positive rate, and achieve the effects of high accuracy, low cost, and improved accuracy

Inactive Publication Date: 2012-08-22
陈国燕
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Technical problem: The purpose of the present invention is to provide a DNA sequencing method based on a primer array chip, which can overcome the shortcomings of the traditional probe array chip hybridization sequencing method using a large number of probes, high cost, and high false positive rate, and can achieve simple operation , low-cost, fas...

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  • DNA sequencing method based on primer array chip

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Embodiment 1

[0021] like figure 1 , fix the 5' ends of a group of primers with known sequences on the substrate at preset positions and arrange them into a primer array chip, and the 3' ends of the primers are 5 bases in total 4 5 = 1024 kinds of all arranged sequences, in which the 3' terminal sequence is a reverse complementary primer, which is fixed at the adjacent position on the substrate, and the middle of the primer is a merging sequence of 5 bases, and its bases are four bases A mixture of bases A, T, C, and G, the 5' end of the primer is 10 bases A, the chip area is less than 3mm×5mm, and can be placed in a 200ul PCR reaction tube.

[0022] Put 4 identical chips into 4 200ul PCR reaction tubes containing four different dideoxynucleotides (ddATP, ddCTP, ddGTP and ddUTP) with fluorescent labels respectively, and the reaction tubes also contain PCR buffer , DNA polymerase and double-stranded DNA to be tested. The double-stranded DNA to be tested is prepared by precipitating with abs...

Embodiment 2

[0026] Fix the 5' ends of a set of primers with known sequences on the substrate at preset positions and arrange them into a primer array chip. The 3' ends of the primers are 6 bases in total 46 = 4096 kinds of all arranged sequences, of which the 3' ends The sequences are reverse complementary primers, which are fixed at adjacent positions on the substrate, and the middle of the primers is a 5-base merger sequence, which is a mixture of four bases A, T, C, and G. The 5' end is 10 base A.

[0027] Add a drop of reaction solution containing four different dideoxynucleotides (ddATP, ddCTP, ddGTP and ddUT), PCR buffer, DNA polymerase and single-stranded DNA to be tested to four identical chips. On the surface of the chip, the single-stranded DNA to be tested is prepared by precipitating the product obtained through asymmetric PCR amplification, washing with 75% ethanol, drying, and dissolving in pure water.

[0028] Put all the above 4 chips into the chip hybridization instrumen...

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Abstract

The invention relates to a DNA sequencing method based on a primer array chip, and is characterized in that the method comprises the following steps: fixing a group of primers with known sequences on a chip, respectively putting four same chips into four reaction systems which contain different dideoxynucleotides with fluorescent tracers, performing annealing hybridization with DNA to be detected to allow the primers to extend 1 nucleotide, detecting the fluorescence signals of the chips to obtain a group of short sequences composed of primer end sequences and extended nucleotides, splicing the short sequences overlapped with each other to obtain a complete sequence of the DNA to be detected.

Description

technical field [0001] The invention belongs to the technical field of DNA sequencing, in particular to a DNA sequencing method based on a primer array chip. technical background [0002] At present, the research on DNA sequencing technology mainly focuses on high-throughput and large-scale sequencing technology. So far, the second-generation sequencing technology of sequencing-by-synthesis and the third-generation single-molecule sequencing technology have been developed. However, the first-generation sequencing technology represented by Sanger terminal termination sequencing still has important applications in conventional biological research. [0003] In 1977, Sanger invented the landmark terminal termination sequencing method, and in the same year A.M.Maxam and W.Gilbert invented the chemical degradation method. The Sanger method has become the mainstream of DNA sequencing so far because it is simple and fast, and has been continuously improved. However, the Sanger met...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈国燕
Owner 陈国燕
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