Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box

Abstract

An identification method and its regent box for recognizing the unit DNA by the allelic ladder of short series-wound repeating serial locus have been opened the invention. The process is: Screening the SRT locus, designing the common reading things for synthesizing special PCR, then to mark the leading things, respectively. Then to extract the DNA of the human, going on the PCR reaction, detecting the part by electrophoresis and purifying the part. Last to connect the product of PCR to the carrier, then getting the clone of the different genes, making the every allele PCR reaction by using the leading thing of fluorescent number, mixing the PCR products of allele for STR locus then adding to the stabilizer. So we can get the allelic ladder. The ladder is the regent box to recognize the DNA. The invention needn't to collect the different gene blood again and again. And the box cost little. It is easy to make, also have good stability.

Description

1. Technical field: [0001] The invention relates to the technical field of genetic engineering, and can be used in the fields of forensic biology, anthropology, genetics and oncology for individual identification, paternity identification, and diagnosis of genetics and tumors, and especially belongs to a genetic engineering method. It is suitable for fluorescent detection of multiple compound STR gene loci, and the STR allele ladder for genotyping, which is used for individual identification DNA detection and identification. 2. Background technology: [0002] Human Genomic DNA has 3×10 9 bp, 10% of which are tandem repeats, known as satellite DNA. According to the length of the repeating unit, it can be divided into large satellites, medium satellites, small satellites and microsatellites. The microsatellite whose repeat unit is only composed of 2-7bp is also called short tandem repeat (short tandem repeat, STR). The number of satellite DNA repeat units in the genomes of ...

AI Technical Summary

Problems solved by technology

[0004] 1. Some STR genetic marker sites have low genetic diversity and polymorphic information in the Chinese population (Mongolian race), and are not suitable for East Asians and Southeast Asians in individual identification DNA testing

[0005] 2. When human genomic DNA is directly selected as the amplification template, due to the different lengths of the alleles, it is easy to have a dominant amplification phenomenon, that is, alleles with shorter lengths are easy to be amplified, while alleles with longer lengths are more likely to be amplified. It is not easy to be amplified, so that the amount of the amplified product is different, and the amount of each allele in the allelic ladder prepared from this is different, and even non-amplified, that is, no amplified product, can not be detected.

[0006] 3. For mass production, it is necessary to frequently select and prepare templates that can represent all allele fragments. Not only the workload is heavy, but also the purity and content of the same template used each time vary, making it difficult to amplify the conditions. standardization

[0007] 4. Human genomic DNA has a large molecular weight and extremely complex structure, which is extremely prone to non-specific amplification and is not suitable for mass production

[0008] 5. Human genomic DNA amplification products are mixed and then amplified again. Since the PCR products are less biologically active than the DNA fragments in bacterial culture and tissue, the non-specific amplification is more serious.

This method is not suitable for mass production

[0009] 6. When the mixture of amplified products is directly used for alleles, it may degrade in a short period of time, making the bands blurred, or the configuration changes to change the electrophoretic migration

Quality changes due to these reasons cannot be used as standard reference objects

[0010] 7. At present, the detection method used in the allelic ladder produced in China is mainly the silver staining method. This method is complicated to operate, has high background, low detection sensitivity and resolution, and is prone to produce uncertain or even misjudged results.

[0011] 8. The prepared allelic ladder is a mixture of alleles of one to three STR sites, and the simultaneous detection of more STR sites is not realized