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Restrictive amplification method

A restriction and restriction enzyme digestion technology, applied in the field of restriction amplification, can solve the problems that hinder the rapid promotion of DNA microarray chip technology, and achieve the effects of accelerating research and industrialization process, uniform length, and improving signal-to-noise ratio

Inactive Publication Date: 2003-12-17
中国人民解放军基因工程研究所
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Problems solved by technology

Therefore, a huge amount of money needs to be invested in the early stage of collecting a large number of target gene fragments, which hinders the rapid promotion of DNA microarray technology to a certain extent.

Method used

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Embodiment 1

[0020] HIV1U26942 DNA is about 9000 bp in length. Analysis of its sequence shows that there are 24 restriction endonuclease Sau3A I recognition sites on the gene. 25 restriction fragments are generated after cutting with this enzyme, of which more than 100 base pairs There are 19 fragments. Analyze the sequence of the first base after GATC at both ends of the digested fragment, and perform PCR reaction with selective primers that extend by 1 base. After electrophoresis recovery, the restriction gene fragment can be quickly prepared. Specific steps are as follows:

[0021] (1) Cut the DNA with restriction endonuclease Sau3A I to generate restriction fragments with "GATC" sticky ends. The enzyme digestion reaction can refer to the following method: take about 1μg HIV1U26942DNA, add 2μl 10× endonuclease buffer, 1μl Sau3A I, add double distilled water (ddH 2 O) To 20μl, react at 37°C for 1 to 2 hours.

[0022] (2) Design two oligonucleotide chains S2P (5′-GAT CCC CCC CCCCCC CCC CCA-3′...

Embodiment 2

[0049] The difference between this example and example 1 is: in step (3), the two ends of the restriction fragment generated by Sau3A I digestion of HIV1U26942 DNA are analyzed for the first two bases after GATC, as shown in example 1. , The fragment was amplified by PCR with UAA and UCC, which was extended by two bases. Therefore, the above 19 fragments can be amplified by 16 PCR reactions with 13 primers that extend two bases, of which 13 reactions amplify a single band, and the other 3 PCR reactions amplify a double band ( TT1610 and TT580, TT553 and TT424, AC456 and AC441). The reaction conditions are the same as above.

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Abstract

A restrictive amplification process for preparing target gene fragment immobilized on DNA microchip includes such steps as using restrictive endonuclease to digest DNA or cDNA designing linker to link said enzyme severed fragment, designing universal primer and selective primer for PCR amplification in different groups, separating and purifying, secondary PCR amplification, purifying, and suspending in solution.

Description

Technical field [0001] The present invention relates to a new method for separating gene fragments, in particular to a restriction amplification method, which can be used to prepare target gene fragments solidified on a DNA microchip. Background technique [0002] Gene chips (or DNA chips) can be divided into two types: DNA chips synthesized in situ and DNA microarays. The latter type of chip is also called a DNA microarray chip. The primary problem in its development is how to collect or prepare meaningful and sufficient DNA fragments. The current preparation methods of target gene fragments solidified on the chip include: ① molecular cloning and PCR (polymerase chain reaction) amplification; this method firstly establishes a cDNA library or a genomic library, and then picks thousands of clones. For identification, PCR amplification, etc., the operation is cumbersome and the technology is complicated; if reverse transcription PCR (RT-PCR) is used to amplify gene fragments, a lar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 马文丽郑文岭李凌
Owner 中国人民解放军基因工程研究所
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