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A kit and extraction method for extracting trace dna

A kit and trace technology, applied in the field of trace DNA detection, can solve the problems of DNA sample loss, less times of tube transfer, cumbersome operation, etc., achieve the effect of reducing trace DNA loss, slow sedimentation speed, and improving extraction efficiency

Active Publication Date: 2020-05-12
广州奇辉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before extracting trace DNA by the Chelex100 method, physical evidence needs to be cleaned, but the cleaning process may lead to further loss of the original trace DNA sample, which increases the difficulty of DNA extraction. At the same time, this method takes a long time to detect and the detection effect is not good.
Although the detection rate of the sample is higher than that of Chelex100, the method of silica beads passing through the column extracts trace DNA, but the operation is cumbersome, the sensitivity is low, and automatic extraction cannot be realized
The magnetic bead method is simpler and more convenient to operate than the silica bead column method, with fewer tube transfers, less contamination and loss, and automatic extraction can be realized. At present, there are magnetic bead methods based on automated workstations on the market to extract trace DNA, but the detection rate less than 50%

Method used

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  • A kit and extraction method for extracting trace dna
  • A kit and extraction method for extracting trace dna
  • A kit and extraction method for extracting trace dna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Internal Control PCR System Verification of Minimum Loading Quantity of Trace DNA

[0056] Experiments were designed to verify the internal control PCR system with the lowest loading amount of trace DNA, and determine the minimum amount of template that can carry out effective PCR reactions.

[0057] Genomic DNA templates of 50ng / μl volunteers were serially diluted to obtain a total of 3000pg, 2000pg, 1000pg, 500pg, 100pg, 50pg, 30pg, 10pg, and 5pg of genomic DNA templates for verification. In order to ensure the accuracy of the experiment, a control experiment was designed: the negative control template was 10mmol / L Tris, which was used to check whether there was nucleic acid contamination in the reaction system; the positive control template was 50ng / μl human genomic DNA, which was purchased from Kangwei Century Human GenomicDNA is used to check whether the amplification ability of the reaction system is normal and whether the sample is abnormal; plus a tota...

Embodiment 2

[0068] Embodiment 2 Reagent component concentration is verified to detection rate

[0069] Design an orthogonal experiment to determine the concentration of NaCl, guanidine isothiocyanate and sodium dodecylsulfonate in the lysate to verify the extraction rate of trace DNA, design an orthogonal experiment with 3 factors and 4 levels; Nacl 0.1mol / L , 0.5mol / L, 1mol / L, 2mol / L; isothiocyanate concentration: 2mol / L, 3mol / L, 4mol / L, 5mol / L; SDS concentration: 1%, 2%, 5%, 10%

[0070] In the experiment, 10^6 cells / ml of cultured cells were used, diluted 10 times, and about 100 cells were extracted from 1 μl of cell suspension

[0071] (1) Add a suspension containing 100 cultured cells to a 1.5ml centrifuge tube;

[0072] (2) To the centrifuge tube with the template added in (1), respectively add the lysate composed of NaCl, guanidine isothiocyanate and sodium dodecylsulfonate at different concentrations according to the orthogonal experiment table, add proteinase K, and pipette mix...

Embodiment 3

[0085] Example 3 micro-cell extraction verification

[0086] The experimental sample is the cultured lung cancer cell line NCI-H1975, the concentration of cultured cells is 10^6 cells / ml, diluted 100 times, and about 10 cells are taken from 1 μl of cell suspension for extraction.

[0087] (1) Add 1 μl suspension containing 10 cultured cells to a 1.5ml centrifuge tube;

[0088] (2) Add 250 μl of lysate and 10 μl of proteinase K;

[0089](3) Using the high-salt environment of the lysate, fully lyse the cells at 2000rpm, 55°C, and 20min to release the genomic DNA of the cells, then add isopropanol of the same volume as the lysate to the lysed mixture and pipette mix;

[0090] (4) Add 7 μl extraction magnetic beads and 10 μl nucleic acid sedimentation aid to the mixed solution obtained in the centrifuge tube, cover the centrifuge tube cap, place the centrifuge tube on a vertical mixer, and mix at room temperature for 10 minutes to allow the magnetic beads and genomic DNA fully ...

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Abstract

The invention belongs to the field of detection of trace DNA and particularly relates to a kit for improving the extraction rate of the trace DNA in applications such as DNA detection, forensic trace DNA detection, trace-sample DNA detection of medical detection and clinical diagnosis, and single-cell DNA detection of tumor cells and germ cells and the like of trace biological detection materials, and a method. The kit contains reagents such as DNA extracting magnetic beads, cracking liquid, protease K, washing liquid, a nucleic-acid settling agent and eluent. The invention also discloses a method for extracting the trace DNA by utilizing the kit. The kit and method disclosed by the invention have the advantages that the detection rate, the accuracy rate and the sensitivity of the trace DNA are improved, simultaneously automatic extraction can be realized, and simultaneous and parallel detection of multiple samples is realized, so that the cost for labor and time is saved.

Description

technical field [0001] The invention belongs to the field of trace DNA detection, and in particular relates to a method for improving DNA detection of trace biological samples, forensic trace DNA detection, medical examination and clinical diagnosis trace sample DNA detection, and single-cell DNA detection of tumor cells and germ cells, etc. The kit and method for the extraction rate of trace DNA. Background technique [0002] Trace DNA refers to DNA in amounts as low as picogram levels. In the field of forensic evidence, DNA detection of trace samples in medical testing and clinical diagnosis, and single-cell DNA detection of tumor cells and germ cells, the number of cells in biological samples detected is small, and the DNA that can be extracted is at a trace level. [0003] In the field investigation of criminal cases, as criminals become more and more aware of anti-reconnaissance, and criminal methods become increasingly concealed and technologically advanced, tradition...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806
Inventor 朱奇王灵敏廖传荣孙继文
Owner 广州奇辉生物科技有限公司
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