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Method for preparing natural AFLP joint

A natural, linker sequence technology, applied in the field of natural AFLP linker preparation, can solve the problems of low linker ligation efficiency, incomplete equal connection of two linkers, difficulty in linker purification, etc.

Inactive Publication Date: 2007-09-19
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to apply modern genetic engineering methods to solve the problems that the joint connection efficiency of the existing chemical synthesis method for making AFLP is low, the purification of the joint is difficult, and the two joints cannot be completely connected in equal amounts.

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  • Method for preparing natural AFLP joint
  • Method for preparing natural AFLP joint

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Embodiment Construction

[0017] 1. First select two complementary single primer DNA sequences, each 60-70bp, the complementary sequences contain the linker core sequence and the two endonucleases required by AFLP, such as the most commonly used enzymes EcoR I and Mse I, single The strands of DNA serve as primers and templates for PCR amplification, and the amplified fragments are ligated into the T vector to obtain the vector pGT-L, which is then sequenced to verify the correctness of the sequence.

[0018] 2. Digest the rice genomic DNA with Spe I and Pst I, tap the rubber to recover a fragment of about 100 bp, similarly use Nco I and Sac II to digest the rice genomic DNA, and tap the rubber to recover a fragment of about 100 bp.

[0019] 3. Digest the pGT-L vector with Spe I and Pst I, recover the large fragment and connect it with the corresponding 100bp small fragment in 2, select different clones for sequencing verification, and select the clone whose insert sequence does not contain any restricti...

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Abstract

A preparing method of natural AF LP joint, selecting plasmid as carrier, using two complent DNA as primers whose sequence containing polymorphisms EcoR I / Mse I, Apa I / Taq I or other polymorphisms which AFLP needed, using PCR method to amplificate joint core area sequence with polymorphisms EcoR I, Mse I, Apa I, Taq I, joining the core area sequence with pGEM-T carrier, and adding different gene DNA passages on two wings of the core area sequence to construct separatedly a joint unit sequence, then connecting different joint unit sequence in series by pBluescript KS carrier to construct DNA carrier containing a plurality of copy joints, introducing natural amplification joint of coliform bacteria, finally cutting joint sequence by EcoR I, Mse I, Apa I, Taq I enzyme, then obtaining DNA with AFLP joint.

Description

technical field [0001] The invention adopts the method of genetic engineering, utilizes the introduction of enzyme cutting sites to construct the recombination vector, naturally propagates the joints from the plasmids, and finally produces the joints for AFLP experiments. Background technique [0002] In the past ten years, a large number of molecular marker technologies have been used in molecular biology research, such as genome analysis, gene cloning, variety identification, and foreign gene introduction. The earliest molecular marker technology used to make maps is RFLP, and a large number of RFLP maps have been constructed so far. However, RFLP technology is time-consuming, it is difficult to analyze a large amount of DNA, and the saturation of the genetic map is low, and the distance between molecular markers is relatively large, which is not conducive to positional cloning. [0003] Since 1990, RAPD, DAF, and AP-PCR technologies have been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/11C12N15/66C12N15/10
Inventor 田大成江海洋唐萍
Owner NANJING UNIV