Preparation method of plasmid deoxyribonucleic acid (DNA) quantitative detection standard

Abstract

The invention discloses a preparation method of a plasmid deoxyribonucleic acid (DNA) quantitative detection standard for the first time. Plasmid DNA is ultrasonically treated into fragments with the lengths of less than 200bp, and is subjected to enzymolysis, a nucleotide marked by a nucleotide standard and an isotope is taken as an internal standard, and mononucleotides are separated and accurately quantified by using high-performance liquid chromatography-mass spectrometry. An optimal condition for the ultrasonic treatment of the plasmid DNA and a condition for the high-performance liquid chromatographic separation of four nucleotides are created. The method is high in precision and accuracy, namely the nucleotide standard is adopted, and dependence on a DNA standard is eliminated, so that a measurement result can be traced back to a nucleotide certified standard material, and the reliability and a traceable source of the measurement result are ensured. The method can be used for absolutely quantifying the plasmid DNA and preparing the plasmid DNA quantitative detection standard.

Description

Technical field: [0001] The invention belongs to the field of biochemical analysis, and relates to a traceable plasmid DNA quantitative method, in particular to a preparation method of a standard product for quantitative detection of plasmid DNA. Background technique [0002] Plasmid DNA is a small (1-200kb) covalent, closed, circular double-stranded DNA molecule free from extrachromosomal, which can autonomously replicate and stably inherit genetic factors. [0003] Quantification of plasmid DNA has a wide range of needs in the field of molecular biology, such as enzyme digestion and ligation operations during cloning library construction, and when sequencing plasmid DNA, in order to obtain good follow-up experimental results, it is necessary to control the concentration of plasmid DNA. Accurate measurement. [0004] Although some of the existing techniques for determining DNA can be used for determining plasmid DNA, all of them have deficiencies. Some require a very larg...

AI Technical Summary

Problems solved by technology

Some require a very large amount of DNA samples, such as the ICP-OES method; some have high costs, such as the consumables of the digital PCR method are too expensive; The following two disadvantages: First, the content value of the DNA standard in the currently commercially available kits is a reference value provided by the manufacturer, which is generally determined by ultraviolet absorption (OD260), and the values ​​of different batches of different manufacturers are different. No relevant uncertainty information and traceability

Second, because PicoGreen has different DNA binding efficiencies of different molecular sizes, if there is a large difference between the quantitative DNA standard and the measured DNA sample molecular size, it will lead to a large deviation in the quantitative results of the sample. Currently, commercial There is only one molecular size DNA standard in all kits, that is, Lambda phage DNA standard (48.5K)

Because the molecular weight of genomic DNA is larger, and the structure is different, it is not circular like plasmid DNA, so the ultrasonic conditions and enzymatic conditions of this method are not suitable for plasmid DNA quantification.

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