Method for preparing fluorescent molecular weight interior label by means of site-specific mutagenesis and enzyme digestion techniques

Abstract

The invention discloses a method for preparing a fluorescent molecular weight interior label by means of site-specific mutagenesis and enzyme digestion techniques. The method comprises the steps of cloning a target DNA fragment A into a plasmid vector to obtain a recombinant plasmid B; conducting site-specific mutagenesis at different length positions of the DNA fragment A with the recombinant plasmid B as the template, and introducing a restriction enzyme cutting site to obtain a corresponding recombinant plasmid C; designing amplification primers F1 and R1 of the DNA fragment A, and conducting fluorescence labeling at the 5' end of each primer; amplifying the recombinant plasmid C by means of the amplification primers F1 and R1, so that an amplification product D is obtained, and purifying the product D; and conducting enzyme digestion on the purified amplification product D by means of restriction enzyme to obtain an enzyme digestion product E, namely the fluorescent molecular weight interior label. By the adoption of the method, molecular weight interior labels of 14 fragments from 50 bp to 500 bp can be prepared, the product does not have impure peaks such as primer peaks, and the process is suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a fluorescent molecular weight internal standard by using site-directed mutagenesis and enzyme cutting techniques. Background technique [0002] The molecular weight internal standard is a series of DNA fragments of different lengths that are electrophoresed in the same channel as the sample during STR detection. During data analysis, the size of the sample DNA can be calibrated by analyzing the relative peak positions of the sample DNA and the molecular weight internal standard. During electrophoresis, the peak time and peak shape of the molecular weight internal standard can also reflect the working status of the electrophoresis instrument. [0003] Chinese patent CN101050474 (disclosure date: 2007.10.10) discloses a method for preparing a molecular weight internal standard and the molecular weight internal standard prepared by this method, and CN 105296473A ...

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Problems solved by technology

However, this method needs to explore the PCR amplification conditions, the amplified products tend to have non-specific bands, and the prepared molecular weight internal standard has poor stability

In the above method, since the length of the primer dimer is similar to the length of the small fragment in the internal standard, it is not easy to purify, so there are primer peaks mixed in, which is easy to cause misjudgment during data analysis, and is not easy to scale production

[0004] At present, there is no literature report on the method of preparing fluorescent molecular weight internal standard by using site-directed mutagenesis and enzyme digestion technology

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