Method for preparing internal reference reagent (InMarker) fluorescent marking DNA molecular weight

Abstract

The invention discloses a method for preparing in marker of fluorescence labeled DNA molecular weight, establishing a template to synthesize the needed fluorescence primer for making PCR reaction, obtaining plasmid of genes of different fragment sizes, preparing amplified product, using fluorescence for detecting, obtaining the in marker of fluorescence labeled DNA molecular weight. The established template of the invention is convenient to identify and the prepared in marker contains special enhance fragments. The manufacturing flow is simple and convenient, short-cycle, simple-operated, and low cost, economical and practical, and suitable for batch-type production.

Description

technical field [0001] The invention relates to genetic engineering, genomics and molecular biology. It applies genetic engineering methods and can provide a reagent for determining the size of DNA fragments, which is not only suitable for ordinary gel electrophoresis, but also suitable for genetic analyzers. This technology can be applied to gene analysis, gene diagnosis and individual identification in the fields of molecular biology, genetics, anthropology, forensic evidence biology and other disciplines. Background technique [0002] The Human Genome Project aims to elucidate the sequence of 3 billion base pairs of the human genome, discover all human genes and find out their positions on chromosomes, and decipher all human genetic information. Studies have found that many traits and diseases of individual organisms are determined by the control of multiple genes, that is, polygenic systems. The study of these traits and lesions should not only focus on the effect of a ...

AI Technical Summary

Problems solved by technology

At present, the DNA molecular weight reference standards used in China are basically obtained by enzymatic digestion. Although the fragment size is also very accurate, they can generally only be used as DNA molecular weight external reference standards. The molecular weight reference standard (marker) reagents used in China are insufficient. Advantages: First, most of them are molecular weight external reference standards, that is, the sample and the molecular weight reference standard are not on the same lane during electrophoresis. Due to the difference in electrophoresis conditions, system errors will be caused

The second is to mass-produce, 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 standardization

The third is to use human genomic DNA as a template, which has a large molecular weight and an extremely complex structure, and is prone to non-specific amplification during amplification, which is not suitable for mass production

Fourth, these reagents are non-fluorescent, and need to add additional dyes. They cannot be used for fluorescence detection, and their sensitivity is low. They can only be applied to ordinary electrophoresis, but not to fluorescence detection instruments such as genetic analyzers. The scope of application is limited.