Chromosome walking method based on PCR (Polymerase Chain Reaction) technology

Abstract

The invention discloses a chromosome walking method based on a PCR (Polymerase Chain Reaction) technology. The chromosome walking method is characterized in that a target DNA (Deoxyribonucleic Acid) sequence is used as a template, two nested specific primers P1 and P2 which extend toward an unknown sequence terminal are designed and compounded on a known sequence, a complementary P3 primer is designed and determined according to the primer P2, firstly the primer P1 is used for guiding the synthesis of a DNA single chain, a terminal tailing enzyme is used for adding more than 15 arbitrary first basic groups to the 3' terminal of the synthesized DNA single chain, and then more than 15 non-complementary second basic groups are added behind the first basic groups; and the tailed DNA single chain is used as a template, and the primers P2 and P3 are used for amplifying unknown genes or regulating element sequences.

Description

technical field [0001] The present invention relates to a method for chromosome walking, more specifically, relates to a method for performing chromosome walking and amplifying unknown gene or regulatory element sequence by using PCR technique and terminal tailing technique. Background technique [0002] Chromosome walking refers to the method and process of starting from the known sequence in the biological genome or genome library to gradually find out the unknown sequence next to it or the target sequence that is linearly related to the known sequence. This technology plays a key role in solving the problems of finding known gene promoters and regulatory elements, studying the boundaries of gene introns and exons, detecting the upstream and downstream sequences of specific sites, and analyzing the insertion sites of foreign genes in the genome. It is an important molecular biology research technique. However, the traditional chromosome walking method based on genomic lib...

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Problems solved by technology

These methods have their own advantages and disadvantages. The ligation-mediated method is limited by the randomness of the restriction site distribution or the ligation efficiency is not high; random or degenerate primers have limited binding ability for some complex templates, and usually require very low annealing Temperature, specificity is not high; although the tailing-mediated method is highly efficient, it does not depend on the template, so the current use of homopolymeric oligonucleotide primers, such as oligo dT, oligo dG, however, oligo dT primers generally require a relatively long time Low annealing temperature, although the annealing temperature of oligo dG primers can be achieved with general specificity primers (Leoni C, Gallerani R, Ceci L R, A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions . 2008, 44: 229-235.), but it is easy to bind to the GC-rich region in the DNA template, so the specificity of such primers is not very strong

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