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DNA (deoxyribonucleic acid) template preparation liquid and DNA template preparation method

A template and blood technology, applied in the field of DNA extraction, can solve the problems of high extraction requirements, cumbersome extraction procedures, and complex components of the preparation solution, and achieve the effect of simple acquisition process, no need for heat treatment, and short time

Active Publication Date: 2020-06-19
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Purpose of the invention: In order to solve the problems of complex components, cumbersome extraction procedures or high extraction requirements of the existing DNA template preparation solution, the first aspect of the present invention provides a DNA template preparation solution, and the second aspect of the present invention provides a DNA template preparation method; the preparation solution of the present invention is especially suitable for animal blood, such as heparin sodium anticoagulant blood of various poultry and mammals

Method used

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  • DNA (deoxyribonucleic acid) template preparation liquid and DNA template preparation method
  • DNA (deoxyribonucleic acid) template preparation liquid and DNA template preparation method
  • DNA (deoxyribonucleic acid) template preparation liquid and DNA template preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Set up 6 tubes of PCR reaction tubes, and take 100 μL of ready-to-use template preparation solution for each tube. The difference between the 6 tubes of ready-to-use template preparation solution is that No. 1 tube of ready-to-use template preparation solution only contains 0.5M NaCl and 15% (v / v) Ethanol, the solvent is water; the ready-to-use template preparation solution for No. 2-6 tubes contains 0.5M NaCl and 15% (v / v) ethanol, and 1-5g / L PVP respectively, and the solvent is water. Add 10 μL rooster heparin sodium anticoagulant blood to each tube. After blowing once with a pipette gun, centrifuge for 1 min in an LX-900 mini centrifuge, and the supernatant can be used as a template for PCR amplification of DNA. The PCR reaction system (50 μL) is shown in Table 1, the nucleotide sequence of the primer CHDF is shown in SEQ ID NO:1, and the nucleotide sequence of the primer CHDR is shown in SEQ ID NO:2.

[0027] Table 1 PCR reaction system

[0028]

[0029] The PC...

Embodiment 2

[0030] Example 2 Preparation of DNA template preparation solution

[0031] Prepare the DNA template preparation solution, including the following components: NaCl, ethanol, PVP, and the solvent is water; wherein the final concentration of NaCl is 0.5M, the volume concentration of ethanol is 15%, and the final concentration of PVP is 5g / L.

Embodiment 3

[0032] Example 3 The ready-to-use template preparation solution is used for rapid sex identification of pigeons

[0033] Take 10 μL of heparin sodium anticoagulant blood from each pigeon, mix them into 100 μL of the ready-to-use template preparation solution prepared in Example 2, blow and suck once with a pipette gun, centrifuge for 1 min in a LX-900 mini centrifuge, and the supernatant is ready Used as PCR amplified DNA template. The PCR reaction system (50 μL) is shown in Table 2, the nucleotide sequence of primer 1 is shown in SEQ ID NO:3, and the nucleotide sequence of primer 2 is shown in SEQ ID NO:4.

[0034] Table 2 PCR reaction system

[0035]

[0036] The PCR reaction conditions were as follows: ①94°C, 5min; ②94°C, 30s; 50°C, 30s, 72°C, 30s, 35 cycles; ③72°C, 5min. PCR products were subjected to 1% agarose electrophoresis, EB staining, and gel imaging system for band typing, see figure 2 . M is DNA molecular weight standard DL2000, - is a negative control, fe...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) template preparation liquid which comprises the following components: a monovalent cation salt, ethanol and water as a solvent, wherein the finalconcentration of the monovalent cation salt is 0.1-0.8M; and the final concentration of the ethanol is 0.5-30%. The invention further discloses a DNA template preparation method which comprises the following steps: (1) mixing anti-freezing blood and the DNA template preparation liquid in a volume ratio of 1:(1-200); and (2) directly centrifuging the mixed liquid obtained in the step (1) or centrifuging the mixed liquid after being left to stand, so as to obtain supernate, namely a DNA template. The instant DNA template preparation liquid disclosed by the invention is free of protease or otherhigh-valent salt ions, simple in obtaining process, free of heating treatment and short in time; and the ratio of the DNA template preparation liquid disclosed by the invention to blood is not strictly required, and DNA templates prepared within a wide range can be all well applied to PCR (polymerase chain reaction) amplification.

Description

technical field [0001] The invention relates to DNA extraction, in particular to a DNA template preparation solution and a DNA template preparation method. Background technique [0002] The use of blood cell DNA for PCR detection has a very broad application prospect, such as gender identification, genetic disease diagnosis, genetic polymorphism analysis, etc. At present, there are roughly three types of methods for obtaining blood genomic DNA templates suitable for PCR amplification: the first type is to extract pure genomic DNA, and the specific acquisition methods include classic phenol-chloroform extraction method, spin column method, etc., and the operation process is cumbersome. , even if the improved spin column method is used, when faced with a large sample size, the entire process of digestion, column passing, washing, and centrifugation will consume a lot of manpower, material and financial resources; the second type is a relatively simple method. That is, the cru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2527/125C12Q2563/137Y02A40/70
Inventor 徐世永刘京鸽陈清茅慧华崔凛李斯旋
Owner JINLING INST OF TECH
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