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Method and reagent for improving rate of extraction of DNAs of castoff cells in case trace sample

A technology of exfoliating cells and extracting efficiency, which is applied in the field of forensic evidence DNA detection, can solve the problems of large influence of personal experience, high price, incomplete length, etc., to reduce dependence on experience, good stability, and reduce peak loss Effect

Active Publication Date: 2011-11-02
深圳柏悦基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for easier and more effective analysis of tiny sample sizes by combining various techniques such as enzyme treatment, chemical reaction or physical separation methods together into one process. It achieves this through an improved technique that combines specific treatments without harming them while still allowing for accurate results. Additionally, there is less dependency upon operators who have expertise compared to current technologies due to its high sensitiveness, rapidness, ease of handling, stable performance over time, nontoxicity, and ability to perform multiple operations simultaneously at once.

Problems solved by technology

This patented technical problem addressed in the patented text relates to finding reliable ways to efficiently analyze traces of criminal suspect specimens during offensive crimes where there could occur many types of harmful agents like bacteria, fungi, yeast, and even if any manmade substances exist around us. Current techniques involve sampling the entirety of the specimen containing minute cellular matter together with its surrounding environment, resulting in difficulties in obtaining high enough nucleic acid levels without compromising the integrity of the original specimen.

Method used

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  • Method and reagent for improving rate of extraction of DNAs of castoff cells in case trace sample
  • Method and reagent for improving rate of extraction of DNAs of castoff cells in case trace sample
  • Method and reagent for improving rate of extraction of DNAs of castoff cells in case trace sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Extraction of trace amounts of DNA from a belt buckle left at the scene of the case.

[0036] (1) Kit preparation

[0037] Prepare a kit for extracting DNA from trace sample materials using magnetic beads, including:

[0038] 1mL silanized magnetic bead solution (0.1mg / μL), a product listed by Wuhan Wawadina Technology Development Co., Ltd. or purchased by Qiagen;

[0039] 100mL lysate, its component is the Tris-HCl buffer solution of pH=8.0;

[0040] 100mL binding solution I, its component volume ratio is 20-50% PEG (molecular weight 8000);

[0041] 20mL combination solution II, its component is ethanol;

[0042] 100mL cleaning solution, its component is ethanol solution with a volume ratio of 80%.

[0043] (2) Use the kit to extract the DNA of the cells shed from the belt buckle left at the scene of the case. The extraction steps are as follows:

[0044] 1) Use the case micro-examination material sticking device to repeatedly stick the belt buckle sever...

Embodiment 2

[0060] Embodiment two control experiments

[0061] Using 1 μL of blood diluted 60 times with normal saline as a simulated sample, replacing the exfoliated cell membrane in Example 1, the amplification and electrophoresis were the same as in Example 1, and a series of control experiments for micro sample extraction were designed as follows:

[0062] In control experiment 1, in the extraction step 1), the film was replaced by 1 μL of simulated sample, and the other steps remained unchanged;

[0063] No protease was added in step 2) of control test 2, and the others were the same as control test 1;

[0064] The incubation temperature in step 3) of control test 3 was less than 70 degrees, and the others were the same as test 1;

[0065] In step 4) of control test 4, ethanol was removed from the binding liquid system, and the others were the same as test 1;

[0066] PEG was removed from the binding liquid system in step 4) of control test 5, and the others were the same as test 1...

Embodiment 3

[0071] The ethanol of embodiment three binding solution II is changed into methyl alcohol and propanol, isopropanol

[0072] The ethanol in the binding solution II in Example 1 is replaced with methyl alcohol, propanol, and isopropanol respectively, and the DNA on the slippers, toothbrushes, and porcelain cups for drinking in the on-site case are extracted respectively with the magnetic bead method;

[0073] Except that 80 μL of binding solution II in the extraction step 3 was replaced with 80 μL of methanol, propanol, and isopropanol, the other operations were the same as in Example 1.

[0074] Figure 10 For the slippers left at the scene of the case, replace the compound STR amplification map of the binding solution II with methanol; Figure 11 For the toothbrush left at the scene of the case, replace the composite STR amplification pattern of the binding solution II with propanol; Figure 12 For the drinking porcelain cup left at the scene of the case, replace the compos...

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Abstract

The invention relates to a method and a reagent for improving the rate of extraction of the DNAs of castoff cells in a case trace sample. Due to the problems of trace amount of case sample, keratinization of cells, multiple polymerase chain reaction (PCR) complex amplification of 16 short tandem repeat (STR) loci and the like, the extraction of the DNAs of castoff cells has long been a difficult point in forensic medicine. According to researches, the combination of pronase digestion, bath at 70 to 100 DEG C and polyethylene glycol(PEG)-ethanol magnetic bead combined system can help to effectively extract trace DNAs and to obtain DNA fragments with a proper length, so that the need of complex amplification of the 16 STR loci in case detection can be satisfied and the success rate of the case detection is improved. The pronase digestion and warm bath allow the castoff cells to release DNAs effectively and ensures the relatively complete structure of the DNAs and the easy combination of the DNAs with the magnetic beads in the PEG-ethanol system. The use of the PEG-ethanol combined system promotes the high-efficiency DNA adsorption of the magnetic beads, ensures the relatively complete structure of the DNA and ensures that DNA fragments with proper length can be obtained to satisfy the need for the complex amplification of the 16 STR loci in case detection.

Description

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Claims

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Application Information

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Owner 深圳柏悦基因科技有限公司
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