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Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid

A technology in magnetic particles and samples, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of not involving purification results, prolonging experimental operation time, and complicated steps, shortening the time for DNA extraction and reducing physical shearing. The effect of cutting and easy operation steps

Active Publication Date: 2012-02-15
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent ZL 200920090335.6 uses nano-scale silicon-coated magnetic microspheres as solid-phase carriers, prepares different lysates, binding solutions, cleaning solutions, etc. to purify the DNA of forensic samples for different samples, but because this method needs to target different samples , configure different lysis, binding, and cleaning systems to purify genomic DNA, and the patented method includes a pre-lysis step, which is cumbersome and complicated, and greatly prolongs the time for experimental operations
Patent ZL 101935646A discloses a method for extracting DNA from trace samples using magnetic beads. This method only describes the purification of genomic DNA from three samples: dried blood spots, saliva, and hair, and fails to involve more samples. Purification result

Method used

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  • Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid
  • Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid
  • Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: The method and effect verification of purifying genomic DNA from blood spots:

[0050] 1. Purification of DNA

[0051] (1) Sample cracking: Utilize a hole puncher to cut the 2 Put the blood spot into a 1.5ml centrifuge tube, add 150μl guanidinium salt lysate successively (the concentration of guanidine salt lysate is 6M, the composition is: 3~6M GuHCl, 3~6M GuSCN, 0.005~0.01M EDTA, 0.005~ 0.01M NaCl, 4-6% Triton X-100 (volume percentage) and 15-80% (volume percentage) alcohol.) and 1.5 μl of DTT (1M), vortex shaker, 95 ° C water bath Fully lyse for 30min.

[0052] (2) Pick out the fully lysed blood spot sample from the carrier.

[0053] (3) Nucleic acid is adsorbed on the solid phase carrier

[0054] Add 10 μl of magnetic particles with a concentration of 30 mg / ml to the lysate obtained in step (2), mix by vortexing, and let stand for 5 minutes to form a magnetic particle-DNA complex.

[0055] (4) Cleaning: take out the centrifuge tube from the magnetic ...

Embodiment 2

[0063] Example 2: The method and effect verification of purifying genomic DNA from oral swabs:

[0064] Oral swab preparation, rinse your mouth with water three times, gently wipe both sides of the mouth with a swab swab, dry in the shade, and set aside.

[0065] 1. Purification of DNA

[0066] When purifying, cut out a thin layer around the oral cavity cotton swab containing oral mucosal cells as an experimental sample. Subsequent steps are the same as in Example 1.

[0067] 2. STR multiplex amplification analysis

[0068] The DNA obtained after purification is carried out to STR composite amplification analysis, and the STR composite amplification analysis method of embodiment 1 is identical, and the result is as follows image 3 As shown, the STR locus is complete, the typing is accurate, and there is no allele loss, unbalanced amplification, etc., indicating that the purification method has high purification efficiency, and the obtained DNA has high purity and good inte...

Embodiment 3

[0069] Embodiment 3: the method and effect verification of purifying genomic DNA from cigarette butt:

[0070] 1. Sample preparation:

[0071] Generally speaking, for cigarette butts with obvious bite marks, we usually cut the part above the bite mark, and for cigarette butts without obvious bite marks, we try to cut the upper end of the cigarette butt, generally cutting a piece with a size of about 2cm×2cm. Shred it as much as possible.

[0072] 2. Purification of DNA

[0073] Step is identical with embodiment 1.

[0074] 3. STR multiplex amplification analysis

[0075] The DNA obtained after purification is carried out to STR composite amplification analysis, and the STR composite amplification analysis method of embodiment 1 is identical, and the result is as follows Figure 4 As shown, the STR locus is complete, the typing is accurate, and there is no allele loss, unbalanced amplification, etc., indicating that the purification method has high purification efficiency, ...

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Abstract

The invention provides a method utilizing magnetic nanocomposite to conveniently, quickly and efficiently purify deoxyribonucleic acid (DNA) from medical samples and other samples containing trace nucleic acid. The method comprises the following steps of: (1) taking a sample to be purified, adding the guanidinium lysis solution with the final concentration of 3-6M and the dithiothreitol with the final concentration of 0.01-0.05M, uniformly mixing, and then carrying out full pyrolysis in a water bath for 5-30min; (2) forming a mixture solution containing magnetic particle-DNA composite; (3) separating the magnetic particle-DNA composite from the mixture solution; (4) cleaning; and (5) eluting to obtain the purified DNA. The method can satisfy the requirement of extracting high-quality DNA from the medical samples, and is applied in downstream polymerase chain reaction (PCR) amplification, submarine thermal reactor (STR) somatotyping and other applications; and the method does not need centrifugation, and has quick and simple operation steps. According to the method, a kit which not only is applicable to manual operation but also can be mated with an automated instrument can be produced, so the medical sample DNA can be obtained through purification.

Description

technical field [0001] The invention relates to a method for purifying DNA by using magnetic nanometer material. Background technique [0002] Nucleic acid extraction is a key step in many molecular biology techniques, and its extraction method is particularly important due to the requirements for purity and integrity. With the rapid development of forensic science, genomics and other disciplines, forensic DNA testing has not only provided the basis for forensic individual identification, but has also become the only high-tech method in criminal technology that can directly identify criminals through the inspection of trace biological evidence. Therefore, choosing a high-performance DNA purification method has become the primary condition for forensic DNA library construction and case analysis. [0003] At present, there are many reports and kits for nucleic acid purification at home and abroad, and they can be roughly divided into two types, one is the phenol-chloroform ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 崔亚丽邓晨杨柳张景阁赵稳操李芳
Owner XIAN GOLDMAG NANOBIOTECH
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