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Enhancing method for polymerase chain reaction

A technology of chain reaction and polymerase, applied in the biological field, can solve the problems of non-specific amplification, poor effect of template enhancement with high GC content, increased product production, etc., and achieve the effect of eliminating inhibition

Active Publication Date: 2013-05-01
CAPITAL NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some non-ionic additives can stabilize the polymerase and prevent the formation of template DNA secondary structure, thereby increasing the production of products, but also causing non-specific amplification
The most common protein-based enhancer, BSA, can improve the stability of DNA polymerase and reduce the adsorption of reactants on the tube wall when amplifying traditional DNA templates, but it is not effective for templates with high GC content.

Method used

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  • Enhancing method for polymerase chain reaction
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  • Enhancing method for polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Polymerase Chain Reaction (PCR) for simple linear templates L and G.

[0028] The template DNA sequence G is a template with high GC content and a thrombin protein recognition interval, and L is a random single-stranded DNA sequence. 100 μL reaction system contains 50 μL Taq enzyme PCR premix buffer, template L or G (600 or 6×10 7molecule), bovine thrombin protein (TB) 2.5μg / mL, forward primer (FP) and reverse primer (RP) 50pmol (Table 1). The PCR reaction conditions were, after pre-melting at 95°C for 30s, 33 rounds of cyclic amplification (denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 30s). The products of the PCR reactions were kept at 4°C until analyzed by gel electrophoresis.

[0029] PCR products were separated by 10% non-denaturing polyacrylamide vertical gel electrophoresis (voltage 150V, time 50min) ( figure 1 ), After staining with gold nucleic acid dye for 10 minutes, the bands were analyzed by taking pictures ...

Embodiment 2

[0032] Example 2: Polymerase Chain Reaction of Random Single-Stranded DNA Libraries.

[0033] In a 50 μL reaction system, 25 μL PCR premix buffer containing Taq enzyme, 30 to 3×10 5 A single-stranded DNA library molecule, bovine thrombin protein 25μg / mL, forward primer (Pool-FP) and reverse primer (Pool-RP) 25pmol (Table 1). The polymerase chain reaction conditions were, after pre-melting at 95°C for 30s, 33 rounds of cyclic amplification (denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 30s). The products of the PCR reactions were kept at 4°C until analyzed by gel electrophoresis.

[0034] figure 2 It is a graph showing that bovine thrombin protein (TB) improves the amplification efficiency of a random single-stranded DNA library under low-concentration conditions. The initial template concentration was 3000 DNA molecules (lanes 2, 3), 300 DNA molecules (lanes 4, 5), and 30 DNA molecules (lanes 6, 7). The sample in lane 1 is a negative contr...

Embodiment 3

[0036] Example 3: Real-time quantitative PCR of a random single-stranded DNA library.

[0037] 20 μL reaction system contains 10 μL PCR premix buffer containing SYBR Green dye, 1.2×10 4 Single-stranded DNA library molecules, bovine thrombin protein (64 μg / mL), forward primer (Pool-FP) and reverse primer (Pool-RP) each 10 pmol (Table 1). The PCR reaction conditions were, after pre-melting at 95°C for 30s, 40 rounds of cyclic amplification (denaturation at 95°C for 20s, annealing at 56°C for 30s, extension at 72°C for 30s). The annealing temperature range is 55°C-95°C.

[0038] image 3 It is a graph showing that the addition of bovine thrombin protein (TB) to real-time quantitative PCR improves the amplification efficiency of a single-stranded random DNA library. 20 μL reaction system contains 1.2×10 4 A single-stranded DNA sequence was added at a concentration of 64 μg / mL of TB.

[0039] Figure 4 is a graph showing the real-time quantitative PCR annealing temperature pr...

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Abstract

The invention relates to an enhancing method for a polymerase chain reaction and belongs to the field of biotechnology. The enhancing method takes bovine thrombin as an additive and is applied to various amplifying templates and amplifying environments so as to obviously increase the efficiency and specificity of PCR (Polymerase Chain Reaction). A defined amount of bovine thrombin is added into a PCR system, so that the amplification efficiency of single-chain DNA template with low template concentration, single-chain random sequence DNA library with low template concentration, HBV (Hepatitis B Virus) in blood serum of a hepatitis B patient and a sex gene in a human whole-genome sample are increased and the forming of primer dimer is restrained. Besides, under the condition of the existence of nano-gold and graphene oxide inhibitors, the bovine thrombin is added for eliminating the inhibiting effect of the inhibitors to the PCR. Compared with the present frequently-used protide PCR additive BSA (Bovine Serum Albumin), the dosage of the bovine thrombin is only 1 / 10 or less than that of the common dosage of BSA.

Description

technical field [0001] The invention relates to a polymerase chain reaction enhancing method, which belongs to the field of biotechnology. Background technique [0002] Polymerase chain reaction (PCR) is an excellent method for in vitro DNA amplification similar to the natural DNA replication process. It mainly consists of three basic reaction steps of denaturation-annealing-extension: ①The double-stranded template DNA is denatured at high temperature and then melted into a single strand; ②Under the action of DNA polymerase, the primer is paired with the complementary sequence of the template DNA single strand; ③DNA The template-primer conjugate uses deoxyribonucleotide (dNTP) as the raw material and the target sequence as the template to synthesize a new semi-reserved replication strand complementary to the template DNA strand according to the principles of base complementary pairing and semi-reserved replication. [0003] PCR technology involves a wide range of applied re...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 娄新徽张颖邹如杏王文洁
Owner CAPITAL NORMAL UNIVERSITY
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