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Pre-amplification method for trace DNA applied in medicolegal expertise

A technology of forensic identification and pre-amplification, which is applied in the fields of forensic evidence biology, criminal investigation, and anthropology. It can solve problems such as difficulties, lost typing, and allelic imbalance, and achieve low-cost effects.

Inactive Publication Date: 2013-11-27
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009]In actual application of the current technology, the detection rate is higher for samples with high DNA content, and the typing is easier, such as blood stains left from the scene, body fluid traces ( Zheng Huifen, Dong Yan et al.2006), and even criminal DNA can be detected on leftover food (Zhang Xiaohong, Tang Jianxin et al.2006), however, for samples with DNA content less than 100pg, allelic imbalance or even loss is likely to occur Typing difficulties (Sun G. et al. 2005, Liu Weiyu and Jin Chunlian 2007, Hellani A. et al. 2004, Ballantyne KN. et al.2007)

Method used

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  • Pre-amplification method for trace DNA applied in medicolegal expertise
  • Pre-amplification method for trace DNA applied in medicolegal expertise
  • Pre-amplification method for trace DNA applied in medicolegal expertise

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Primer Mix amplifies the method of 100pg, 50pg and 10pg DNA template under 4 gradients, and specific content is as follows:

[0051] 1. The preparation of the primer Mix mixture, the primer Mix mixture is a mixture of 36 primers from primer1-primer36, prepare 4 gradient primer Mix mixtures, the concentration gradient of the mixture is as follows:

[0052] Primer Mix 1: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.1uM)

[0053] Primer Mix 2: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.05uM)

[0054] Primer Mix 3: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.01uM)

[0055] Primer Mix 4: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.005uM)

[0056] 2. Add the followi...

Embodiment 2

[0066] Embodiment 2: the method for amplifying 100pg, 50pg and 10pg DNA using random primer 9N, primer1 and primer2 double primers, primer1 single primer and Pmier Mix, the specific contents are as follows:

[0067] 1. Random primer 9N amplifies 100pg, 50pg and 10pg DNA, the amplification system is as follows:

[0068] 9N 3ul

[0069] 10×Reaction Buffer 2ul

[0070] 10 (or 50 or 100) pg / ul DNA 1ul

[0071] wxya 2 O 7ul

[0072] Pre-denature at 98°C for 10 minutes, take out the deformed solution, place it on ice quickly for 15 minutes, then add the following reagents to it, and finally amplify at 33°C for 16 hours, 65°C for 15 minutes; 4°C∞;

[0073] Each 2.5mM dNTP 4ul

[0074] 0.5% (w / v) BSA 2ul

[0075] 5000U Phi29 1ul

[0076] figure 2 In "9N", the initial template amount of 1-2 is 100pg, 3-4 is 50pg, 5-6 is 10pg, and 7 is a negative control; no amplification band is shown in the figure.

[0077] 2. Primer 1 (final concentration 3uM) single primer amplifies 100pg,...

Embodiment 3

[0108] Embodiment 3: Applied to the forensic identification trace DNA pre-amplification method, using Pmier Mix to amplify the method of 100pg, 50pg and 10pg DNA, the specific content is as follows:

[0109] (1) Put 36 kinds of primers from primer1-primer36 into a single PCR tube and mix evenly. The final concentration of primer1 and primer2 is 1uM, and the final concentration of each primer from primer3-primer36 is 0.05μM. Divide into three groups;

[0110] (2) Add 10 μL of 10× reaction buffer, 42 μL of double distilled water and 1 μL of 10, 50 or 100 pg / ul trace DNA template to each group of mixed solutions;

[0111] (3) Place the mixture in step (2) in a PCR instrument for pre-denaturation at 98°C for 5 minutes;

[0112] (4) Take out the denatured solution, place it on ice for 10 minutes, then add 6 μl of dNTP, 3 μl of 0.5% BSA, and 2uL of 5000U Phi29 DNA polymerase, mix well and put it back into the PCR instrument for amplification. 2.5mM, PCR amplification conditions are...

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Abstract

The invention discloses a pre-amplification method for trace DNA applied in medicolegal expertise. The method aims at amplifying the trace DNA to obtain high-concentricity DNA template so as to facilitate following medicolegal expertise. The method mainly comprises the steps of exploring the optimum concentration and proportion volume of combined primers; and the amplification conditions are further optimized, the trace DNA aims for being amplified with high efficiency and high fidelity, so that the possibility for the next legal medical expert is provided for determining individuals.

Description

technical field [0001] The invention relates to an efficient pre-amplification method applied to forensic identification of trace DNA, which belongs to the fields of forensic evidence biology, anthropology, criminal investigation and the like. Background technique [0002] In the current forensic identification, DNA amplification technology tends to be mature, but when the amount of template is less than 100pg, it is difficult to detect by conventional methods. At the same time, fingerprints are very important in criminal investigation, but some feature points of the crime scene are incomplete. Incomplete fingerprints often hinder forensic identification. Usually there are very few exfoliated cells in fingerprints. Due to the small number of cells and the low total amount of DNA, it is difficult to become a DNA sample. [0003] In actual cases, there are often fingerprints left by the suspect or the person responsible for the incident at the crime scene. Conventional fingerp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 罗瑛朱晖唐文如柳海涛李安史斌蔡海强
Owner KUNMING UNIV OF SCI & TECH
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