Preparation method of universal short tandem repeat (STR) allele ladder

Abstract

The invention provides a preparation method of a universal short tandem repeat (STR) allele ladder. The preparation method comprises the following steps of (1) designing and synthesizing a primer anda plasmid according to an STR locus; (2) using the primer to perform a PCR amplification reaction by using the plasmid as an amplification formwork; and (3) performing purification on PCR amplification products, and performing appropriate dilution so as to obtain the STR allele ladder. According to the preparation method disclosed by the invention, only one plasmid needs to be synthesized, the PCRreaction needs to be performed only once, and after the PCR products are subjected to purification and dilution, the allele ladder of the corresponding STR locus can be obtained, so that the preparation technological process of the allele ladder of the STR locus is greatly simplified, and labor cost,, time cost and economic cost for preparation are saved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a general short tandem repeat (STR) allele ladder (Allele Ladder) and a kit thereof. Background technique [0002] A genetic marker called short tandem repeat (STR) exists in human and various genomes. STR is widely used in the research of genetics, forensic science, oncology, etc., especially those STR genetic markers with high polymorphism in the population. Such as Dib C and his colleagues in 1996 (Nature.1996 Mar 14; 380(6570):152-4.PMID:8600387), Broman KW is equal to 1998 (Am J Hum Genet.1998 Sep; 63(3):861- 9.PMID:9718341), Kong A et al. in 2002 (Nat Genet.2002 Jul; 31(3):241-7.PMID:12053178) used 5236, 8325, and 5136 STR genetic markers respectively for high-resolution Genetic mapping of the human genome. In the field of forensic science, STR markers have become standard genetic markers for individual identification and parentage dete...

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Problems solved by technology

[0018] (3) Although the method of gene cloning is a solution to the need to continuously collect different DNA samples, for a typing kit containing 20 different STR loci, it is often necessary to complete hundreds of alleles Gene cloning requires huge workload and cost

However, the problem with this solution is that first, the cost of whole gene synthesis is high, and second, the technical difficulty for whole gene synthesis of repeat structure is high, especially for STR genes with long repeat domains, such as FGA

[0020] (5) Even after the cloning of all alleles is obtained by gene cloning or whole gene synthesis, a more complicated process is how to mix hundreds of alleles equally

When carrying out plasmid mixing, the slight difference of different allelic plasmid templates also can lead to the significant deviation of each allelic yield in the PCR product obtained after PCR amplification (as seen in the accompanying drawing of CN105886497A)

[0021] (6) However, if a single allele clone is used for saturated PCR amplification, the PCR products of different alleles are purified, quantified and remixed, although the significant deviation of the content of different allele fragments in the allele ladder can be reduced, but due to this The workload in the process is huge, and the impact of this mixing process on the production environment (mainly the possibility of contamination of PCR products) is also a problem that cannot be ignored for a long time

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