Concentrated PCR buffer and its application
By using concentrated PCR buffer, the problems of low amplification efficiency and poor specificity of low-concentration DNA test materials on forensic site were solved, and efficient and sensitive DNA detection effects were achieved, which was suitable for forensic genetic testing.
Patent Information
- Application Number
- CN202510021262.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art is difficult to effectively process low-concentration and high-degradation DNA test materials for forensic on-site, and the existing PCR buffers are inefficient and have poor specificity in low-concentration DNA amplification.
Concentrated PCR buffer was used to increase the initial addition amount of DNA template and enhance the amplification specificity.
It improves the detection rate and amplification efficiency of low-concentration DNA samples, enhances the anti-inhibition ability, ensures the specificity and sensitivity of the amplification results, and is suitable for forensic identification and kinship identification.
Smart Images
Figure CN119776503A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of forensic genetics detection, and relates to a detection buffer for low-concentration DNA samples at a forensic scene, in particular to a concentrated PCR buffer and an application thereof. Background Art
[0002] As the second generation of genetic markers after restriction fragment length polymorphism, short tandem repeat (STR) has been widely used in forensic evidence research and identification since the 1980s and 1990s, and is currently one of the most widely used genetic markers. Compared with other genetic markers, STR loci have small fragments and are easy to amplify, which is more suitable for trace and degraded samples. In addition, multiple STR loci can be amplified simultaneously, which has the advantages of rapidity, efficiency, accuracy, sensitivity, and large amount of information. It is widely used in individual identification and kinship identification in forensic evidence.
[0003] The basic components of the PCR composite amplification reaction system include primers, dNTPs, DNA polymerase, Mg 2+ and template. Primers are oligonucleotides containing 15-30 bases that can bind to the flanking sequences of the target region in the template DNA; dNTPs are composed of four basic nucleotides: dATP, dCTP, dGTP and dTTP, which are the raw materials for the synthesis of new DNA chains; DNA polymerase is the catalyst for PCR reactions; Mg 2+ It is a cofactor of DNA polymerase, which helps promote the binding of dNTP; the template is the nucleic acid molecule containing the target region. During the PCR reaction, DNA polymerase catalyzes the extension from the 3' end of the primer. 2+ Under the action of dNTPs, new DNA chains are synthesized with dNTPs as raw materials. When performing PCR reactions, a buffer is usually added. The components in the buffer can provide a suitable chemical reaction environment for DNA polymerase, such as a stable pH range (pH = 8.0-9.5) and K + wait.
[0004] Forensic samples, such as bloodstains, semen, saliva, body fluids, hair, etc., often have low DNA concentration and small fragment length. In order to improve the detection rate of the above samples, the existing technology often uses a method of simultaneous composite amplification of multiple STR loci. However, there is no better solution for dealing with more extreme low-concentration samples. Summary of the invention
[0005] Technical problem to be solved: In order to overcome the deficiencies of the prior art, for low-concentration and highly degraded DNA samples at the forensic scene, the template addition amount of the samples can be increased on the one hand, and the amplification specificity of samples from different sources can be improved on the other hand. In view of this, the present invention provides a concentrated PCR buffer and its application.
[0006] Technical solution: concentrated PCR buffer, the concentrated PCR buffer comprising the following components: 100mM-300mM Tris-HCl, 50mM-250mM KCl, 0.02-0.05% gelatin by mass fraction, 5mM-25mM dNTPs, 2mM-20mM MgCl2, 2U / μL Taq DNA polymerase, 5%-25% glycerol by volume fraction, and 0.1%-10% polyethylene glycol 6000 by mass fraction.
[0007] Preferably, the concentrated PCR buffer comprises an additive, and the additive is at least one of polyvinyl pyrrolidone, Tween 20 and bovine serum albumin.
[0008] Preferably, in the concentrated PCR buffer, the mass fraction of polyvinyl pyrrolidone is 0.1%-10%, the volume fraction of Tween 20 is 5%-15%, and the mass concentration of bovine serum albumin is 1 mg / mL-35 mg / mL.
[0009] Preferably, the molecular weight of the polyvinyl pyrrolidone is 55,000.
[0010] Bovine serum albumin can effectively improve the anti-inhibition ability and protect DNA polymerase. Preferably, the additive is bovine serum albumin; or bovine serum albumin and Tween 20; or bovine serum albumin, Tween 20 and polyvinyl pyrrolidone 55000.
[0011] Preferably, the concentrated PCR buffer comprises the following components: 175mM Tris-HCl, 200mM KCl, 0.035% gelatin by mass, 15mM dNTPs, 12mM MgCl2, 2U / μL Taq DNA polymerase, 15% glycerol by volume, 2% polyethylene glycol 6000 by volume, 10% Tween 20 by volume, 10mg / mL bovine serum albumin and 1% polyvinylpyrrolidone 55000 by mass.
[0012] Application of any of the above concentrated PCR buffers to increase the initial addition amount of low-concentration DNA templates in a PCR reaction system.
[0013] Use of any of the above concentrated PCR buffers in improving the specificity of PCR reactions.
[0014] Use of any of the above concentrated PCR buffers in improving the detection rate of DNA fragments.
[0015] Use of any of the above concentrated PCR buffers in forensic identification.
[0016] The working principle of the concentrated PCR buffer of the present invention is that polyethylene glycol can increase the viscosity of the reaction system, reduce the reaction between primers, reduce the probability of primer dimer formation, further increase the amount of primers, and improve the amplification efficiency. Polyvinyl pyrrolidone is a polymer chemical with good solubility, film-forming properties, biocompatibility and chemical stability, which can effectively reduce the conformational stability of the DNA template, making the DNA template easy to melt in the denaturation stage of PCR, thereby improving the PCR amplification efficiency and specificity.
[0017] Beneficial effects: (1) When the concentrated PCR buffer of the present invention detects low-concentration DNA samples at the forensic scene, it can increase the amount of initial DNA template, improve the detection rate, enhance the anti-inhibition ability, and improve the adaptability of the test material; (2) In the same amplification system as the traditional PCR buffer, the system using the concentrated PCR buffer of the present invention can add a larger volume of test samples, and has good amplification efficiency for both extracted samples and direct amplification samples; (3) The concentrated PCR buffer has the advantages of strong specificity, high sensitivity, and accurate typing results, and can fully meet the needs of actual case inspection, DNA database construction, and kinship identification. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The present invention cooperates with the AGCU EX28CS fluorescence detection kit to amplify and extract samples;
[0019] Figure 2 Extract samples for amplification of the original system of the AGCU EX28CS fluorescence detection kit;
[0020] Figure 3 The present invention cooperates with the AGCU EX28CS fluorescence detection kit to amplify the direct amplification sample;
[0021] Figure 4 Amplify the direct amplification samples for the original system of the AGCU EX28CS fluorescence detection kit;
[0022] Figure 5 The present invention cooperates with the AGCU EX28CS fluorescence detection kit to amplify low-concentration samples, wherein the sample is added to the entire water replenishment volume in the amplification system;
[0023] Figure 6 The low-concentration sample was amplified by the original system of the AGCU EX28CS fluorescence detection kit, wherein the sample was added to the entire water volume in the amplification system. DETAILED DESCRIPTION
[0024] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0025] Example 1
[0026] In this example, the AGCU EX28CS fluorescence detection kit of Wuxi Zhongde Meilian Biotechnology Co., Ltd. was used as the experimental control, and the concentrated PCR buffer described in this example was used to replace the original system as the experimental group.
[0027] The components and final concentrations of the concentrated PCR buffer described in this example are as follows: 175 mM Tris-HCl, 200 mM KCl, 0.035% gelatin, 15 mM dNTPs, 12 mM MgCl2, 2 U / μL Taq DNA polymerase, 15% glycerol, 2% polyethylene glycol 6000, 10% Tween 20, 10 mg / mL bovine serum albumin, and 1% polyvinylpyrrolidone 55000.
[0028] The steps of using the buffer solution of the test group and the control group to detect the actual sample are as follows:
[0029] 1. Collect genomic DNA
[0030] The sample sources used in forensic identification, paternity testing or DNA family tree construction include human genomic DNA extracted by Chelex-100 method, magnetic bead extraction method or organic extraction method; or human blood or oral cells collected by any carrier such as filter paper, FTA card, cotton swab or gauze without extraction. The sources of samples include human blood, blood stains, semen, saliva, body fluids, hair, muscle or tissue organs.
[0031] 2. PCR amplification
[0032] A. Amplification system is shown in Table 1:
[0033] Table 1 PCR amplification system
[0034]
[0035] B. PCR Amplification Procedure
[0036] Place the PCR tube on a PCR instrument and perform amplification using the program in Table 2. After amplification, store the sample away from light.
[0037] Table 2 PCR amplification program
[0038]
[0039] C. Genetic analyzer fluorescence detection of amplified products
[0040] The loading mixture was composed of deionized formamide and the molecular weight internal standard AGCU Marker SIZ-500 in the system [(0.5 μl AGCU Marker SIZ-500 × (number of injections) + (12 μl deionized formamide) × (number of injections)]. 12.5 μl of the loading mixture was mixed with 1 μl of the amplified product or the allelic ladder (Wuxi Sino-German Meilian Biotechnology Co., Ltd.) to avoid bubbles. Denature at 95°C for 3 minutes, ice bath for 3 minutes, and perform electrophoresis detection using a genetic analyzer as soon as possible.
[0041] 3. Type analysis
[0042] Analyze the data collected by the Genetic Analyzer assay using fragment analysis software.
[0043] The results are as follows: The amplification results of the sample extracted by the present invention in combination with the AGCU EX28CS fluorescence detection kit are shown in Figure 1 , the results of direct amplification samples are shown in Figure 3 The same sample was amplified using the original system of the AGCU EX28CS fluorescence detection kit. The results of the amplified and extracted samples are shown in Figure 2 , the results of direct amplification samples are shown in Figure 4 The results of using the present invention in combination with the AGCU EX28CS fluorescence detection kit to amplify low-concentration samples are shown in Figure 5 (The sample is added to the entire replenishment volume). The results of amplifying low-concentration samples using the original system of the AGCU EX28CS fluorescence detection kit are shown in Figure 6 (The sample is added to the entire water volume). Comparing the results of the two, the present invention can increase the amount of template added by 50% under the same amplification system when used in conjunction with the AGCU EX28CS fluorescence detection kit, thereby improving the detection effect of low-concentration templates. When amplifying direct amplification samples, the overall detection effect is better when the present invention is used in conjunction with the AGCU EX28CS fluorescence detection kit.
Claims
1. A concentrated PCR buffer, characterized in that The concentrated PCR buffer comprises the following components: 100mM-300mM Tris-HCl, 50mM-250mM KCl, 0.02%-0.05% gelatin by mass fraction, 5mM-25mM dNTPs, 2mM-20mM MgCl2, 2U / μL Taq DNA polymerase, 5%-25% glycerol by volume fraction, and 0.1%-10% polyethylene glycol 6000 by mass fraction.
2. The concentrated PCR buffer according to claim 1, characterized in that The concentrated PCR buffer comprises an additive, and the additive is at least one of polyvinyl pyrrolidone, Tween 20 and bovine serum albumin.
3. The concentrated PCR buffer according to claim 2, characterized in that In the concentrated PCR buffer, the mass fraction of polyvinyl pyrrolidone is 0.1%-10%, the volume fraction of Tween 20 is 5%-15%, and the mass concentration of bovine serum albumin is 1mg / mL-35mg / mL.
4. The concentrated PCR buffer according to claim 2, characterized in that The polyvinyl pyrrolidone is polyvinyl pyrrolidone 55000.
5. The concentrated PCR buffer according to claim 2, characterized in that The additive is bovine serum albumin; or Bovine serum albumin and Tween 20; or bovine serum albumin, Tween 20 and polyvinylpyrrolidone 55000.
6. The concentrated PCR buffer according to claim 1, characterized in that The concentrated PCR buffer comprises the following components: 175 mM Tris-HCl, 200 mM KCl, 0.035% gelatin by mass fraction, 15 mM dNTPs, 12 mM MgCl2, 2 U / μL Taq DNA polymerase, 15% glycerol by volume fraction, 2% polyethylene glycol 6000 by volume fraction, 10% Tween 20 by volume fraction, 10 mg / mL bovine serum albumin and 1% polyvinyl pyrrolidone 55000 by mass fraction.
7. Use of the concentrated PCR buffer according to any one of claims 1 to 6 for increasing the initial addition amount of low-concentration DNA template in a PCR reaction system.
8. Use of the concentrated PCR buffer according to any one of claims 1 to 6 in improving the specificity of PCR reactions.
9. Use of the concentrated PCR buffer according to any one of claims 1 to 6 in improving the detection rate of DNA fragments.
10. Use of the concentrated PCR buffer according to any one of claims 1 to 6 in forensic identification.