An additive for improving PCR specificity and its application

Abstract

The present invention relates to the field of molecular biology technology, and in particular to an additive for improving PCR specificity and its application. The PCR additive containing trivalent metal ions of the present invention can significantly inhibit the generation of coating tailing bands, significantly improve the PCR specificity, effectively eliminate the coating tailing bands appearing in the gel at an appropriate concentration, and optimize the PCR product into a single target band; the PCR additive of the present invention has the advantages of strong stability, low economic cost, simple operation method, etc., and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to an additive for improving PCR specificity and application thereof. Background Art

[0002] The polymerase chain reaction (PCR) is capable of performing genetic testing on samples containing only a few cells. Because PCR technology can amplify DNA in test samples to a high degree, it holds significant advantages in biomedicine, clinical testing, and the detection of pathogenic microorganisms in food. However, factors such as insufficient sample purity, high GC content in the template DNA, and inappropriate primer design can result in the appearance of nonspecific or smearing bands in the PCR product, or even the failure to amplify the target band, compromising experimental results.

[0003] Currently, nanoparticles such as dimethyl sulfoxide, glycerol, bovine serum albumin, betaine, and gold, silver, and magnetite (Fe₃O₄) nanoparticles have been reported to improve PCR yield and specificity to a certain extent. However, some substances may pose potential risks to human health and environmental safety. Therefore, the search for stable, economical, and non-toxic PCR additives to address PCR nonspecificity remains crucial.

[0004] In existing patents, bovine thrombin protein, mannosylglycerol, diglyceride phosphate, and ectoine are used as PCR additives to enhance PCR efficiency. However, to date, there have been no reports on PCR additives containing trivalent metal ions that can improve PCR specificity. Summary of the Invention

[0005] The purpose of the present invention is to find a stable, economical and non-toxic PCR additive for improving PCR specificity, and a PCR additive containing trivalent metal ions is designed.

[0006] The PCR additive containing trivalent metal ions provided by the present invention is used to improve PCR specificity, primarily by significantly suppressing or even eliminating the generation of nonspecific bands (primarily smearing bands) in electrophoretic gels. The principle of the invention is as follows: when both long and short DNA fragments are present in the PCR components, the addition of metal ions to the PCR components can significantly alter the spatial conformation of the long DNA fragments (e.g., condense them into clumps) compared to the short DNA fragments, resulting in preferential inhibition of the amplification of the long fragments. Therefore, based on this principle, the addition of trivalent metal ions to the PCR system can eliminate nonspecific bands associated with large fragments.

[0007] In the first aspect of the present invention, there is provided the use of trivalent metal ions in the preparation of PCR additives.

[0008] The mechanism by which the PCR additive significantly improves PCR specificity is based on the fact that trivalent metal ions preferentially inhibit the amplification of long fragments in the PCR system, thereby improving the smear tailing phenomenon that occurs during the PCR amplification process.

[0009] Further, the trivalent metal ions are gold ions (Au

[0013] ,

[0012] , ,

[0011] , , ,

[0015] , ,

[0014] ), chromium ions (Cr 3+ ), aluminum ions (Al 3+ ), and complex ions containing the above ions, such as AuCl4 - and so on.

[0010] Further, the trivalent metal ions are derived from various forms of metal salt compounds or their solutions containing metal ions of the above valence states, such as gold chloride (AuCl3), gold hydroxide (Au(OH)3), chloroauric acid (HAuCl4) and its hydrated compounds (such as HAuCl4·3H2O), triclopyridine gold or pyridine gold trichloride (C5H5AuCl3N), aluminum chloride (AlCl3), chromium chloride (CrCl3) and its hydrated compounds (such as CrCl3·6H2O), etc.

[0011] Further, when the trivalent metal ions are added to the PCR system, the final concentration range of the trivalent metal ions is 0.1 - 200 μM (micromoles per liter).

[0012] Depending on the concentration of the template DNA, the expected length of the PCR product, and the number of PCR amplification cycles, the optimal concentration of the trivalent metal ions required is different.

[0013] In the second aspect of the present invention, there is provided a PCR additive containing trivalent metal ions. The PCR additive is a buffer solution (such as a 10 mM Tris-HCl solution) that dissolves a substance containing trivalent metal ions (salts or complexes) to obtain a buffer (such as Tris-HCl) solution containing trivalent metal ions and their various complex and hydrated ions.

[0014] Further, the pH value of the PCR additive is 6 < pH < 7. The PCR reaction system is a weakly alkaline system. If the pH of the PCR additive is too low, it may affect the pH value of the PCR reaction system. If the pH of the additive is higher than 7, the metal ions will form hydroxide precipitates, affecting the use effect.

[0015] Preferably, the PCR additive containing trivalent metal ions of the present invention can be prepared by the following method: dissolving a trivalent metal ion salt in a prepared Tris-HCl solution with a concentration of 10 mM to obtain a solution containing a trivalent metal ion salt dissolved in the Tris-HCl solution, and adjusting the solution to 6 <pH<7。

[0016] A third aspect of the present invention provides a use of the above-mentioned PCR additive containing trivalent metal ions in preparing a PCR reaction system.

[0017] In a fourth aspect, the present invention provides a method for improving PCR specificity, comprising adding, in addition to the essential components for PCR amplification, a PCR additive containing trivalent metal ions when preparing a PCR reaction system, wherein the final concentration of the trivalent metal ions in the PCR system is 0.1-200 μM.

[0018] Furthermore, the optimal concentration of the required trivalent metal ions varies depending on the concentration of the template DNA, the expected PCR product length, and the number of PCR amplification cycles.

[0019] Furthermore, the method for improving PCR specificity includes but is not limited to the following steps:

[0020] (A) Preparation of PCR reaction system: Add the necessary components for PCR amplification, such as template, primers, enzyme, dNTPs, and an appropriate amount of PCR additive containing trivalent metal ions, to a PCR reaction tube.

[0021] (B) PCR reaction program: Set the temperature and time for pre-denaturation, denaturation, annealing, and extension; the annealing temperature and extension time should be adjusted appropriately according to the gene to be amplified.

[0022] (C) Detection of PCR amplification results: PCR products were detected using agarose gel electrophoresis.

[0023] The advantages of the present invention are:

[0024] 1. The present invention designs a PCR additive containing trivalent metal ions for improving the specificity of PCR and inhibiting the generation of nonspecific bands (mainly coating tailing bands) that appear in electrophoresis gels. When long and short fragments are present in the PCR components, after adding trivalent metal ions to the PCR components, the trivalent metal ions bound to the long fragments will cause conformational changes in the DNA template, resulting in its preferential inhibition during the subsequent amplification process.

[0025] 2. Experiments have shown that the PCR additive containing trivalent metal ions designed in the present invention can significantly inhibit the occurrence of coating tailing phenomenon, significantly improve the specificity of PCR, and effectively inhibit the generation of nonspecific bands in the gel at an appropriate concentration, thereby optimizing the PCR product into a single target band.

[0026] 3. The PCR additive designed in the present invention has the advantages of strong stability, low economic cost, simple operation method, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Contains Au 3+ PCR additives to eliminate nonspecific bands in PCR. M represents BM2000 DNA marker, 1 represents no Au-containing 3+ The control group of PCR additives, 2-4 means that the PCR system was added with Au 3+ PCR additive, Au 3+ The final concentrations were 10, 20, and 30 μM, respectively.

[0028] Figure 2 .Contains Cr 3+ The PCR additives are used to eliminate non-specific bands in PCR. M represents BM2000 DNA marker, and 1 represents no Cr-containing 3+ The control group of PCR additives, 2-7 means that the PCR system was added with Cr 3+ PCR additive, Cr 3+ The final concentrations were 10, 20, 30, 40, 50, and 60 μM, respectively.

[0029] Figure 3 Contains Al 3+ PCR additives to eliminate nonspecific bands in PCR. M represents BM2000 DNA marker, 1 represents no addition of Al 3+ The control group of PCR additives, 2-4 means that the PCR system was added with Al 3+ PCR additives, Al 3+ The final concentrations were 7.5, 15, and 30 μM, respectively. DETAILED DESCRIPTION

[0030] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0031] Example 1: Containing Au 3+ PCR additives to improve PCR specificity

[0032] 1) Upstream primer: 5′-GGCTTCGGTCCCTTCTGT-3′ (SEQ ID No. 1), downstream primer: 5′-CACCACCTGTTCAAACTCTGC-3′ (SEQ ID No. 2). Template (Lambda DNA), DNA polymerase, dNTP and other conventional reagents were purchased from a biological company.

[0033] 2) PCR system is as follows: 1.25U / 25μL Ex Taq DNA polymerase, 20mM Tris-HCl (pH 8.9), 20mM KCl, 2mM MgCl2, dNTPs and primers (0.04μM). 3+ The PCR additive was added to the PCR system at a final concentration of 30 μM.

[0034] 3) PCR amplification procedure: Initial denaturation at 95°C for 2 min. 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Finally, extension at 72°C for 5 min. PCR products were stored at 4°C.

[0035] 4) Detect the PCR amplification results by agarose gel electrophoresis.

[0036] PCR amplification results are as follows Figure 1 As shown, no Au 3+ The PCR product in the control group of PCR additives containing Au was a coating tailing band. 3+ In the experimental group containing PCR additives, nonspecific bands were suppressed, and the PCR product could be optimized into a single target band at an appropriate concentration. 3+ The PCR additive can indeed eliminate the coating tailing phenomenon and improve the specificity of PCR.

[0037] Example 2: Containing Cr 3+ PCR additives to improve PCR specificity

[0038] 1) Upstream primer: 5′-GGCTTCGGTCCCTTCTGT-3′ (SEQ ID No. 1), downstream primer: 5′-CACCACCTGTTCAAACTCTGC-3′ (SEQ ID No. 2). Template (Lambda DNA), DNA polymerase, dNTP and other conventional reagents were purchased from a biological company.

[0039] 2) PCR system is as follows: 1.25U / 25μL Ex Taq DNA polymerase, 20mM Tris-HCl (pH8.9), 20mM KCl, 2mM MgCl2, dNTPs and primers (0.04μM).3+ The PCR additive was added to the PCR system at a final concentration of 40 μM.

[0040] 3) PCR amplification procedure: Initial denaturation at 95°C for 2 min. 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Finally, extension at 72°C for 5 min. PCR products were stored at 4°C.

[0041] 4) Detect the PCR amplification results by agarose gel electrophoresis.

[0042] PCR amplification results are as follows Figure 2 As shown, no Cr-containing 3+ The PCR product in the control group of PCR additives containing Cr was a coating tailing band. 3+ The nonspecific bands in the experimental group of PCR additives were significantly improved, and the PCR product could be optimized into a single target band at an appropriate concentration, indicating that the PCR product containing Cr 3+ The PCR additive can indeed inhibit the spreading tailing phenomenon and improve the specificity of PCR.

[0043] Example 3: Containing Al 3+ PCR additives to improve PCR specificity

[0044] 1) Upstream primer: 5′-GGCTTCGGTCCCTTCTGT-3′ (SEQ ID No. 1), downstream primer: 5′-CACCACCTGTTCAAACTCTGC-3′ (SEQ ID No. 2). Template (Lambda DNA), DNA polymerase, dNTP and other conventional reagents were purchased from a biological company.

[0045] 2) PCR system is as follows: 1.25U / 25μL Ex Taq DNA polymerase, 20mM Tris-HCl (pH8.9), 20mM KCl, 2mM MgCl2, dNTPs and primers (0.04μM). 3+ The PCR additive was added to the PCR system at a final concentration of 15 μM.

[0046] 3) PCR amplification procedure: Initial denaturation at 95°C for 2 min. 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Finally, extension at 72°C for 5 min. PCR products were stored at 4°C.

[0047] 4) Detect the PCR amplification results by agarose gel electrophoresis.

[0048] PCR amplification results are as follows Figure 3 As shown, no Al3+ In the control group of PCR additives containing Al 3+ The non-specific bands in the experimental group of PCR additives were obviously inhibited, indicating that the Al 3 + The PCR additive can indeed inhibit the spreading tailing phenomenon and improve the specificity of PCR.

[0049] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence Listing <110> Shanghai Taidao Testing Technology Co., Ltd. <120> An additive for improving PCR specificity and its application <130> / <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial <400> 1 ggcttcggtc ccttctgt 18 <210> 2 <211> twenty one <212> DNA <213> Artificial <400> 2 caccacctgt tcaaactctg c 21

Claims

1. Use of trivalent metal ions in the preparation of a PCR additive for improving PCR specificity; the trivalent metal ions are gold ions or chromium ions, and complex ion forms containing gold ions or chromium ions; the trivalent metal ions are derived from various metal salt compounds or their hydrated compounds containing gold ions or chromium ions; when the trivalent metal ions are added to the PCR system, the final concentration of gold ions is 10 - 30 μM, and the final concentration of chromium ions is 10 - 60 μM; the pH value of the PCR additive is 6 < pH < 7.

2. A method for improving PCR specificity, characterized in that: When preparing the PCR reaction system, in addition to adding the essential components for PCR amplification, a PCR additive containing trivalent metal ions is also added; the trivalent metal ions are gold ions or chromium ions, and complex ion forms containing gold ions or chromium ions; the trivalent metal ions are derived from various metal salt compounds or their hydrated compounds containing gold ions or chromium ions; among them, the final concentration of gold ions in the PCR system is 10 - 30 μM, and the final concentration of chromium ions in the PCR system is 10 - 60 μM; the pH value of the PCR additive is 6 < pH < 7.

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