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Method for cloning end of target gene 5' and special kit thereof

A kit and gene technology, applied in the field of cloning the 5' end of the target gene, can solve the problems of high degeneracy and randomness of degenerate primers, and achieve the effects of simple and feasible experimental steps, reduced experimental costs, and saved time

Inactive Publication Date: 2010-09-08
INST OF BOTANY CHINESE ACAD OF SCI
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Problems solved by technology

The flanking sequence of known partial sequence information can also be obtained by thermal asymmetric polymerase chain reaction (Thermal Asymmetric Interlaced PCR, TAIL-PCR), but the degenerate primers used in this method have a large degree of degeneracy. When the end of the gene is cloned, the randomness is greater

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  • Method for cloning end of target gene 5' and special kit thereof

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Embodiment 1

[0073] Cloning of the 5' end of chalcone synthase gene (PcCHS2 gene) in embodiment 1, Polygonum cuspidatum

[0074] According to the partial gene sequence information of Polygonum cuspidatum gene PcCHS2 that has been obtained, design gene-specific (GSP) primers, GSP1, GSP2 and GSP3, the nucleotide sequences of GSP1, GSP2 and GSP3 are as follows:

[0075] GSP 1: 5'-CTCAACCGACTTCTTCCTCATCT-3';

[0076] GSP2: 5'-GCGAGTCCCAATCACTCACATT-3';

[0077] GSP3: 5'-GAAGCAGATAGCGGTTATTTCG-3'.

[0078] PCR reaction program:

[0079] The first round of PCR reaction system:

[0080] 50μL reaction system: 250μM dNTP, 10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mMMgCl 2 , TDP1 primer 20μM, GSP1 primer 20μM, 0.5mg Polygonum cuspidatum genomic DNA as template.

[0081] The first round of PCR cycle parameters are:

[0082] Pre-denaturation at 98°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 2.5 minutes, cycle 6 times;

[0083] Denatura...

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Abstract

The invention discloses a method for cloning the end of target gene 5'and a special kit thereof. The method comprises the following steps of: respectively combining a degenerate primer TDP which is designed according to a TATA-box conserved sequence with gene specific primers GSP1, GSP2 and GSP3 into 3 pairs of primers; and amplifying the end of the gene 5' by means of semi-nested PCR with the 3 pairs of primers, wherein the Tm values of the gene specific primers GSP1, GSP2 and GSP3 are higher than the Tm value of the TDP degenerate primer at 3-5 DEG C, and the combination positions between the gene specific primers GSP1, GSP2 and GSP3 and the gene are sequentially near to the end of the gene 5'. The method for cloning the end of target gene 5' does not need to use expensive RNA extraction reagent, RACE extraction reagent and the like, thereby reducing the experiment cost.

Description

technical field [0001] The invention relates to a method for cloning the 5' end of an objective gene and a special kit thereof. Background technique [0002] In modern biological research, cloning full-length genes is an indispensable step for in-depth study of gene structure and function. Through library screening or homologous cloning, only partial DNA sequence information of the gene is often obtained. To obtain the full length of the gene, it is necessary to obtain the 5' end sequence information and the 3' end sequence information of the gene. Currently, the commonly used method for cloning the 3' end and 5' end of a gene is Rapid Amplification of cDNA End (RACE). When using the cDNA terminal rapid amplification method to clone the 3' end of the gene, the 3' end of the mRNA has a special polyadenine tag (polyA), which is easy to clone; the mRNA is very easy to degrade, and the cloning of the 5' end of the gene requires more involvement The steps of mRNA manipulation, ...

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 王红郭艳武马兰青刘本叶李国凤叶和春
Owner INST OF BOTANY CHINESE ACAD OF SCI
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