STR gene marker based PCR primer and kit for identifying donor DNA chimeric ratio, application thereof and chimera detection method

Abstract

The invention discloses an STR gene marker based PCR primer and kit for identifying donor DNA chimeric ratio, application thereof and a chimera detection method. The detection method comprises the steps of: a) extracting DNA from a sample to serve as a template; b) setting the claim 1 required annealing temperature of the PCR primer, and marking the PCR primer; c) conducting PCR amplification on the marked PCR primer to obtain an amplification product; and d) analyzing the fragment size of the amplification product, and determining the gene marker having differences. According to the chimera detection method involved in the embodiment of the invention, the PCR primer and the fluorescence labeled DNA fragment analysis technologies are organically combined to create a complete of chimeric ratio detection analysis system with the advantages of high donor-recipient differentiation rate, simple and quick operation, and accurate result analysis. The method overcomes the subjective influence in traditional methods, and has the characteristics of high resolution, accurate result, simple and quick operation, cleanness and no toxicity.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, relates to a PCR primer, a kit and a detection method for its application for identifying donor DNA chimerism ratio based on STR gene markers. Background technique [0002] At present, the evaluation of engraftment after hematopoietic stem cell transplantation mainly relies on the detection of donor chimerism. Early methods for detecting donor chimerism include phenotype detection, sex chromosome karyotype analysis, and HLA typing. These methods can obtain objective evidence of engraftment in some cases, but they all have certain application limitations. With the development of molecular biology, the application of PCR technology to amplify and analyze the genotypes of donors and recipients can facilitate individual identification. Gene polymorphism molecular markers used for amplification analysis include restriction fragment length polymorphism (RFLP), minisatellite ...

AI Technical Summary

Problems solved by technology

However, this method is complicated to operate, time-consuming, with obvious subjective error and low accuracy.

Commonly used STR primers must use different amplification programs for preliminary typing of donors and recipients due to different annealing temperatures, which cannot rationally allocate resources and time

The preparation of polyacrylamide gel uses more reagents, the procedure is cumbersome and it is difficult to ensure the same quality of gel preparation every time

The electrophoresis process takes 2-3 hours, and the heat generated by the long-term electrophoresis will affect the electrophoresis quality of the gel due to uneven heating

During the gel staining process, the difference in the silver staining and constant volume reagent used each time, and the temperature change will cause the difference in the background to affect the judgment

Due to the high resolution of polyacrylamide gel electrophoresis, the non-specific bands generated by amplification often have a huge impact on the real amplified bands

Optical density quantification belongs to relative quantification, and the error is large

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