A new method for preparing short tandem repeat allelic ladders

Abstract

The invention discloses a novel method for preparing a short tandem repeat sequence (Short Tandem Repeats, STR) allelic ladder. This method mainly designs and synthesizes PCR amplification primers and plasmids based on the STR information on the locus, uses the synthesized plasmid as a PCR amplification template, and amplifies all alleles of the same locus in one tube, and then proceeds according to the difference. The prepared short tandem repeat sequence allelic ladder is obtained after group amplification, purification and appropriate dilution. The present invention achieves the purpose of obtaining a well-balanced allelic ladder with fewer times of PCR reactions, can ensure the balance of the product amount of each allelic fragment without special adjustment, and saves the production cost in the preparation process , labor costs and time costs, greatly improving work efficiency.

Description

technical field [0001] The invention belongs to the fields of gene detection technology and genetic engineering, and more specifically relates to a method for preparing a novel short tandem repeat sequence allelic ladder. Background technique [0002] Short tandem repeats (Short Tandem Repeats, STR) is a core sequence composed of 2-6bp repeating units. It is a DNA fragment widely distributed in the human genome, and length polymorphism is mainly caused by changes in the copy number of the core repeat sequence. , follow the Mendelian law of inheritance, and heredity is stable. It is a commonly used genetic marker at present, and is widely used in genetics, forensic science, clinical medicine, oncology, individual identification in genetic diagnosis, paternity test, tumor diagnosis and other fields. [0003] In forensic individual identification and paternity identification, the most commonly used method of STR is to first amplify the STR locus by PCR, then separate fragments ...

AI Technical Summary

Problems solved by technology

[0010] Using the above scheme to prepare an allelic ladder and extract DNA from human nucleated cells for amplification, the process of extracting samples is not only cumbersome and requires a lot of work, but also some alleles are difficult to collect completely, and a large number of samples are required. Screening, this process is time-consuming and laborious, not suitable for mass production

Moreover, in order to continue the production, DNA samples need to be collected all the time, and the quality difference of different templates will cause the quality difference of alleles in different batches

Using sample DNA or plasmid as a template to amplify a single allele and then purify and quantitatively mix the products requires hundreds of amplifications of two or three hundred fragments, which is too much work and the difference between different alleles Significant differences will appear after small differences are amplified, and it is difficult to carry out accurate quantitative mixing, which is difficult and cumbersome, resulting in poor balance between different alleles and high environmental requirements for the production workshop

Although amplifying all alleles at the same time can greatly reduce the workload, shorter fragments can be amplified better during the amplification process, but the products of long fragments are not easy to amplify in equal amounts, resulting in differences in the amount of amplified products. Larger, even a small number of alleles have no amplification products, resulting in poor balance of alleles in the final allele ladder

[0011] In order to solve the shortcomings of the above method for preparing allelic ladders, patent CN110229871A discloses "a general method for preparing allelic ladders of short tandem repeat sequences". Although the technical solution provided by this invention is generally applicable to all loci, but It is possible to change the sequence of the allele, resulting in the position of the core repeat domain of the allele fragment and the position in the DNA fragment are different, and its specificity and specificity are poor; and the efficiency of the primer and the process of random combination are considered , the alleles of the locus may not all be amplified in a balanced manner

At present, there is a lack of an allelic ladder preparation method that can improve work efficiency and ensure the accuracy and completeness of allelic sequences as well as good balance and specificity

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