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Nucleic acid sequencing process based on micro array chip

A microarray chip and nucleic acid sequencing technology, which is applied in the field of biomedical nucleic acid array chip applications, can solve the problems of high nucleic acid sequencing cost, low preparation cost, and high sequencing throughput, and achieve the effects of simple implementation, high throughput, and mature technology

Inactive Publication Date: 2007-03-21
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Technical problem: Aiming at the current high cost and slow speed of nucleic acid sequencing, the present invention proposes a DNA sequencing method combining traditional Sanger (Sanger) sequencing method and nucleic acid microarray chip technology, that is, based on microarray chip Nucleic acid sequencing method, which has high sequencing throughput, low preparation cost, and simple method, can be used for direct sequencing detection of target nucleic acids, and has very important application prospects and use value

Method used

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  • Nucleic acid sequencing process based on micro array chip
  • Nucleic acid sequencing process based on micro array chip
  • Nucleic acid sequencing process based on micro array chip

Examples

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Effect test

Embodiment 1

[0035] Example 1. Nucleic acid microarray chip prepared from immobilized PCR products for sequencing

[0036] 1. Preparation of Universal Primer PCR Products

[0037] Taking amplification as an example, to measure the nucleic acid sequence of the hepatitis B virus DNA fragment 78-257, the specific method is:

[0038] 5'...GGCATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCCTTTTTG

[0039] CCTTCTGACTTCTTTCCGTCGGTACGAGATCTTAGATACCGCCTCAGCTCTATATCGGGAAGCCTTAGAGTCTCCTGAACATTGCTCACCTCACCATACTGCACTCAAGCAAGCAATT...3', design a pair of specific amplification primers, P1:

[0040] 5’...NH 2 -TTTTTTTTTTCCGTACCTGTAACTAG...3',

[0041] P2: 3'...CTCAAGCAAGCAATT GCATGGCCACGGGCGGAC ...5'. The italic part is the specific primer sequence, and the bold part is the general sequence, that is, the 5' end of all PCR downstream primers has this fragment. The HBV 78-257 fragment is amplified in a conventional PCR amplification system by using the pair of primers.

[0042] 2. Preparation of ge...

Embodiment 2

[0047] Example 2. Nucleic acid microarray chip prepared by fixed rolling circle amplification technology for sequencing

[0048] 1. Apply the rolling circle amplification technique to prepare microarray chips of single-molecule multi-copy DNA fragments of the whole genome (as shown in Figure 1-III).

[0049]Fragmentation of genomic DNA: as shown in Figure 6, ①Use an ultrasonic instrument to break the genomic DNA into fragments of 100-500 bp at a certain intensity, and take 1 μl of the product to gel in 1.5% agarose Gel electrophoresis for 30 minutes to detect the effect of ultrasonic fragmentation of genomic DNA; ②Take a certain amount of ultrasonic product, add an appropriate amount of mung bean nuclease system, and react at 30°C for 30-60 minutes, because this enzyme can remove single-stranded 3′ or 5′ overhangs blunt end of all DNA fragments, and then inactivate mung bean nuclease at 80°C for 15 minutes; ③Add an appropriate amount of Lambada DNA nuclease system to the react...

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Abstract

The nucleic acid sequencing process based on nucleic acid micro array chip is for sequencing genome and detecting gene transcription and expression spectrum of some specific species, and detecting various genome DNA modifying spectrum. Different DNA segments to be sequenced are fixed onto one solid chip to form nucleic acid micro array, and each of the DNA segments is made to contain the same common sequence as the complementary sequence of the sequencing primer. After crossing the common sequencing primer and the chip, some mixed nucleotide solution with certain ratio of fluorescent marker ddNTP and dNTP is injected to the chip to terminate on-chip base reaction with a micro flow controller by means of Sanger end terminating process. Once the termination is finished, one fluorescent signal is obtained through scanning to obtain the base extending information in different sample points on the chip. All the sequencing maps are finally superposed to obtain the complete nucleic acid segment information.

Description

technical field [0001] The present invention relates to a nucleic acid sequencing method based on a microarray chip, especially a method for performing genome sequencing of a specific species, detection of gene transcription and expression profiles, and various genomic DNA (deoxyribonucleotide) modification profiles (such as DNA The detection of methylation profile) belongs to the technical field of biomedical nucleic acid array chip application. Background technique [0002] The human genome (sequencing) project has been completed, and the post-genome project has entered into implementation. Especially after the completion of the Human Genome Project, relevant nucleic acid sequence data has grown exponentially, and more and more genome sequences of animals, plants, and microorganisms have been determined, providing a wealth of information resources for comparative genomics research. How to analyze and compare a large amount of biomolecular information, find common molecula...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 白云飞肖鹏峰涂景吕华陆祖宏
Owner SOUTHEAST UNIV
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