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

A quantitative detection and standard product technology, applied in the field of biochemical analysis, can solve the problems of unsuitable plasmid DNA, large amount of DNA samples, and expensive consumables, etc.

Active Publication Date: 2013-04-24
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 ef

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1 Quantification of transgenic plasmid pNK603 by ultrasound-isotope dilution mass spectrometry

[0125] 1. Materials and methods:

[0126] 1. The transgenic plasmid pNK603, with a size of 3307bp, was developed by the China Institute of Metrology and was obtained by connecting two exogenous gene fragments (zSSIIb and NK603) to the vector pEASY-T3. The specific qualitative extension primer sequences are shown in Table 7.

[0127] Table 7 Primer Sequence

[0128]

[0129] 2. Ultrasonic treatment

[0130] A sonic focusing ultrasonic breaker (Covaris S2) was used, and the processing conditions were intensity: 5; time 25 min; sample volume 100 μL; Duty Cycle: 10%, Cycle per Burst: 200, and temperature: 4°C. DNA concentration range: 30-40ng / μl.

[0131] 3. Preparation of dNMP standard solution

[0132] The specific process of preparation is the same as above, the difference is that the standard solution needs to be prepared according to the approximate concentr...

Embodiment 2

[0173] Example 2 Quantification of Plasmid pBAZS by Ultrasonic-isotope Dilution Mass Spectrometry

[0174] 1. Materials and methods:

[0175] 1. Plasmid pBAZS, with a size of 5206bp, was developed by the China Institute of Metrology and was obtained by ligating four exogenous gene fragments on the vector pEASY-T3 (see the table below for the primers used for amplification); see Table 14 for the specific amplification primer sequences.

[0176] Table 14 Primer Sequence

[0177]

[0178] 2. Ultrasonic treatment

[0179] A sonic focusing ultrasonic breaker is used, and the processing conditions are intensity: 5; duty cycle: 20%; cycle / burst: 200; time 25min; sample volume 0.1mL; DNA concentration range: about 30ng / μl.

[0180] 3. Preparation of dNMP standard solution

[0181] The dNMP standard substance was dried in an oven according to the instructions for use, dAMP, dCMP, and dGMP were dried at 80°C for 4 hours, and dTMP was dried at 40°C for 4 hours. Accurately weigh a...

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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...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/02
Inventor 董莲华孟盈王晶傅博强高运华张玲
Owner NAT INST OF METROLOGY CHINA
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