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Method for extracting purified DNA from human exfoliative cells

A technology of exfoliated cells and human body, applied in the field of DNA extraction and purification, which can solve the problems of inability to meet the requirements of DNA extraction and purification, insufficient DNA purity, etc.

Inactive Publication Date: 2011-09-14
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 purified DNA from human exfoliative cells
  • Method for extracting purified DNA from human exfoliative cells
  • Method for extracting purified DNA from human exfoliative cells

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: the pH is 7.4, the solvent is water, and the solute is the following final concentration: 10mM Tris-HCl, 0.5g / 100ml sodium dodecylsulfonate (SDS), 1mM EDTA and 50mM NaCl aqueous solution , 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 concentrations: 4M guan...

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 purified DNA from human exfoliative cells. The method comprises the following steps: 1) pretreating: contacting the human exfoliative cells with pretreating lysate to remove the solid matters adhering to the biological cells to obtain coarse lysate; 2) nucleic acid adsorbing: mixing the coarse lysate obtained in the step 1) with lysis integrated liquid and magnetic bead suspension to form a solution system containing a magnetic nano microsphere-DNA compound and collecting the magnetic nano microsphere-DNA compound in the solution system; and 3) washing: washing the magnetic nano microsphere-DNA compound obtained in the step 2) with washing liquid I and washing liquid II respectively and then dissolving out the DNA with eluent. The efficiency of extracting DNA by the method can reach 90% and the extracted DNA can be applied to such downstream analysis operations as STR (short tandem repeat) multiplex amplification, DNA sequencing, DNA quantifying and the like.

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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