Method for extracting and purifying spittle DNA

A saliva and solution technology, which is applied in the field of DNA extraction and purification, can solve the problems of not meeting the requirements of DNA extraction and purification, and insufficient DNA purity.

Inactive Publication Date: 2011-10-19
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 spittle DNA
  • Method for extracting and purifying spittle DNA
  • Method for extracting and purifying spittle DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Extraction and purification of DNA in a trace amount of blood and verification of its effect

[0068] 1. Extraction and purification of DNA in trace amounts of blood and STR multiplex amplification analysis

[0069] 1. Reagents

[0070] The reagents are as follows:

[0071] 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.

[0072]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.

[0073] Washing solution I: the solvent is water, and the solute is the following final...

Embodiment 2

[0111] Example 2, Extraction and Purification of DNA in Purified Blood Spots and Validation of Effects

[0112] 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.

[0113] 1. Extraction and purification of DNA from blood spots

[0114] 1. Reagents

[0115] The reagents are as follows:

[0116] 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.

[0117] 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.

[0118] Washing solution I: ...

Embodiment 3

[0138] Example 3, Extraction and Purification of DNA in Purified Blood Spots and Validation of Effects

[0139] 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.

[0140] 1. Extraction and purification of DNA

[0141] 1. Reagents

[0142] The reagents are as follows:

[0143] 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.

[0144] 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 spittle DNA, which comprises the following: 1) a step of pre-treatment, which is to contact the spittle with pretreatment lysis solution, remove solid matters adhered onto cells and obtain coarse lysis solution; 2) a step of nucleic acid absorption, which is to mix the coarse lysis solution obtained by the step 1) with lysis binding solution and magnetic bead suspension to form a solution system containing magnetic nano microsphere and DNA composite and is to collect the magnetic nano microsphere and DNA composite from the solutionsystem; and 3) a step of washing, which is to wash the magnetic nano microsphere and DNA composite obtained by the step 2) with washing liquid I and washing liquid II in turn, and dissolve out DNA byusing eluent. When the method provided by the invention is used, the DNA extraction efficiency may reach 90 percent, the extracted DNA can be used in short tandem repeat (STR) composite amplification, DNA sequencing, DNA quantification and other downstream analysis and operation.

Description

[0001] This application is a divisional application of the following invention: the filing date is August 5, 2009, the application number is 200910090336.0, and the invention name is a method for extracting and purifying DNA. technical field [0002] The invention relates to the field of biotechnology, in particular to a method for extracting and purifying DNA. Background technique [0003] 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. [0004] Various nucleic acid purification methods that cu...

Claims

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Application Information

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