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Method for extracting and purifying DNA

A lysing solution and pretreatment technology, applied in the field of DNA extraction and purification, can solve the problems of insufficient DNA purity and inability to meet the requirements of DNA extraction and purification.

Inactive Publication Date: 2009-12-30
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purity of DNA extracted by this method is not enough to meet the requirements of DNA extraction and purification of trace DNA biological samples.

Method used

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  • Method for extracting and purifying DNA
  • Method for extracting and purifying DNA
  • Method for extracting and purifying DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Extraction and purification of DNA in a trace amount of blood and verification of its effect

[0067] 1. Extraction and purification of DNA in trace amounts of blood and STR multiplex amplification analysis

[0068] 1. Reagents

[0069] The reagents are as follows:

[0070] Pretreatment lysate: pH is 7.4, and solvent is water, and solute is the material of following final concentration: 10mM Tris-HCl, the sodium dodecylsulfonate (SDS) of 0.5g / 100ml, the NaCl aqueous solution of the EDTA of 1mM and 50mM, 0.5 mg / ml proteinase K.

[0071] Lysis binding solution: pH is 6.4, solvent is water, and solute is the material of following final concentration: the Tris-HCl of 50mM, the guanidine isothiocyanate of 3M, the Triton X-100 of 1% (volume percentage), 0.5g / 100ml 3-(3-cholaminopropyl)-dimethylamino-1-propanesulfonic acid (CHAPS), 10 mM EDTA and 15% (volume percent) ethanol.

[0072] Washing solution I: the solvent is water, and the solute is the following fina...

Embodiment 2

[0110] Example 2, Extraction and Purification of DNA in Purified Blood Spots and Validation of Effects

[0111] A 3×3mm blood spot on the FTA card was used as a biological sample, and the DNA in the sample was extracted and purified.

[0112] 1. Extraction and purification of DNA from blood spots

[0113] 1. Reagents

[0114] The reagents are as follows:

[0115] Pretreatment lysate: pH is 8.0, the solvent is water, and the solute is the substance of the following final concentration: 50mM Tris-HCl, 3g / 100ml sodium dodecylsulfonate (SDS), 50mM CDTA and 100mM NaCl aqueous solution, 0.2 mg / ml proteinase K.

[0116] Lysis binding solution: pH is 7.0, solvent is water, and solute is the material of following final concentration: the Tris-HCl of 100mM, the guanidine isothiocyanate of 5M, the Triton X-100 of 5% (volume percent), 2g / 100ml 3-(3-cholaminopropyl)-dimethylamino-1-propanesulfonic acid (CHAPS), 50 mM EDTA and 20% (volume percent) ethanol.

[0117] Washing solution I: ...

Embodiment 3

[0137] Example 3, Extraction and Purification of DNA in Purified Blood Spots and Validation of Effects

[0138] A sample in which 2 μl of anticoagulated blood was dropped on a cotton swab was used as a biological sample, and DNA in the sample was extracted and purified.

[0139] 1. Extraction and purification of DNA

[0140] 1. Reagents

[0141] The reagents are as follows:

[0142] Pretreatment lysate: pH is 8.5, and solvent is water, and solute is the material of following final concentration: 100mM phosphate (100mM NaH 2 PO 4 , 20mM NaCl, pH 8.2), 5.0g / 100ml of sodium lauryl sarcosine (SLS), 100mM of EGTA and 150mM of NaCl in water, 5.0mg / ml of proteinase K.

[0143] Lysis binding liquid: pH is 7.4, and solvent is water, and solute is the material of following final concentration: the Tris-HCl of 150mM, the guanidine thiocyanate of 8M, the Tween 20 of 10% (volume percentage), the 3- (3-cholaminopropyl)-dimethylamino-1-propanesulfonic acid (CHAPS), 100 mM EGTA and 30% (...

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Abstract

The invention discloses a method for extracting and purifying DNA, comprising the following steps: 1) pre-treatment: a biological sample is in contact with pre-treated lysis solution, and solid substance adhering to biological cells is removed to obtain rude lysis solution; 2) nucleic acid adsorption: the rude lysis solution obtained in step 1 is mixed with lyse combined liquid and magnetic bead suspension to form a solution system containing magnetic nanosphere-DNA composite body, and the magnetic nanosphere-DNA composite body in the solution system is collected; 3) washing: the magnetic nanosphere-DNA composite body obtained in step 2 is successively washed with a cleaning solution I and a cleaning solution II, and then DNA is dissolved out by spent regenerant. The method provided by the invention has 90% of the DNA extracting efficiency, the extracted DNA can be used in down stream analysis operation, such as STR multiplex amplification, DNA sequencing, DNA quantitation and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting and purifying DNA. Background technique [0002] DNA separation and purification is a very important operation in molecular biology experiments. The quality of nucleic acid is directly related to the smooth progress of subsequent operations. Advances in biology, forensics, and genomics have intensified the need for sophisticated methods for obtaining nucleic acids from various biological samples. For example, DNA provides a wide range of information about genetic origins and genetic polymorphisms. This information can be used in the practice of forensic genetic identification. [0003] Various nucleic acid purification methods that currently exist can be divided into the following two categories: liquid phase purification and solid phase purification. In liquid phase purification, the nucleic acid is maintained in a liquid state, and impurities are removed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04
Inventor 叶健周云彪赵兴春
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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