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Method for improving polymerase chain reaction amplification effect by utilizing uridine

A technology of chain reaction and polymerase, which is applied in the field of polymerase chain reaction amplification, can solve the problems of inconvenient application and large difference in effect, and achieve the effect of improving specificity, obvious effect, and easy-to-master optimization method

Active Publication Date: 2011-09-14
山东大正医疗器械股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these additives have their own limitations, and the effects vary greatly in different situations, which brings inconvenience to the application.

Method used

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  • Method for improving polymerase chain reaction amplification effect by utilizing uridine
  • Method for improving polymerase chain reaction amplification effect by utilizing uridine
  • Method for improving polymerase chain reaction amplification effect by utilizing uridine

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Experimental program
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preparation example Construction

[0024] 1. Preparation of uridine (Uridine) solution:

[0025] Uridine, also known as Uridine, 1-β-D-Ribofuranosyl uracil, has a molecular formula of C9H12N2O6 and a molecular weight of 244.20. The uridine solid powder can be a commercially available product, which belongs to the prior art and will not be described in detail. The concentration of the uridine solution is as follows: solutions with different concentrations can be prepared according to needs, and the unit of the concentration is mass-volume ratio (W / V), and the range is 40-50 mg / mL. The general recommended concentration is 40mg / mL. Taking the uridine solution with a concentration of (W / V) 40mg / mL as an example, first accurately weigh 40mg of uridine powder, put it in a sterilized 1.5mL small centrifuge tube, and slowly add the sterilized solution with a pipette. water to dissolve, and finally dilute to 1 mL.

[0026] 2. Sterilization of uridine solution: All solutions need to be sterilized at high temperature (...

Embodiment 1

[0040] Synergistic effect of uridine on general PCR (non-high GC):

[0041] 1) Preparation of uridine solution: 40mg / ml;

[0042] 2) Sterilization of uridine solution: high temperature sterilization (121°C, 30min);

[0043] 3) Configuration of PCR reaction system:

[0044] Configure the system in thin-walled PCR tubes in the following order and dosage:

[0045] 10X PCR Buffer 1.2μL

[0046] dNTPs (25mM) 0.1μL

[0047] Template (100ng / ul) 0.2μL

[0048] Primer 1 (2.0μM) 0.75μL

[0049] Primer 2 (2.0μM) 0.75μL

[0050] Mg 2+ (25mM) 1.2μL

[0051] Taq (5U / μL) 0.2μL

[0052] Uridine (40mg / ml) or H 2 O 7.6 μL

[0053]

Primer 1

Primer 2

Amplified length

1

TTTATGGTTGTTGCCCCTCTCCTA

AGAAGAAAAAGCCTGAGCTTGGT

881

[0054]Among them, the template is human Genome DNA, which is extracted by our laboratory. Taq enzyme and PCR buffer were purchased from Beijing Sanbo Yuanzhi Bioengineering Company. Configure 2 tubes of PCR system in...

Embodiment 2

[0063] Synergistic effect of uridine on high GC sequence PCR:

[0064] 1) Preparation of uridine solution: 40mg / ml;

[0065] 2) Sterilization of uridine solution: high temperature sterilization (121°C, 30min);

[0066] 3) Configuration of PCR reaction system:

[0067] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0068] 10X PCR Buffer 1.2μL

[0069] dNTPs (25mM) 0.1μL

[0070] Template (100ng / ul) 0.2μL

[0071] Primer 1 (2.0μM) 0.8μL

[0072] Primer 2 (2.0μM) 0.8μL

[0073] Mg 2+ (25mM) 1.2μL

[0074] Taq (5U / μL) 0.2μL

[0075] Uridine (40mg / ml) or H 2 O 7.5 μL

[0076]

[0077] Among them, the template is human Genome DNA, which is extracted by our laboratory. Taq enzyme and PCR buffer were purchased from Beijing Sanbo Yuanzhi Bioengineering Company. A total of 12 tubes of PCR system were configured, and 6 pairs of primers were used, numbered 1, 2, 3, 4, 5, 6; the numbers of uridine added were 1', 2', 3', 4', 5', 6 '. Th...

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Abstract

The invention discloses a method for improving a polymerase chain reaction (PCR) amplification effect by utilizing uridine, and relates to the biotechnical field. The method is realized by adding a certain amount of uridine into the system on the basis of the conventional PCR amplification method. The method has a remarkable effect of optimizing the amplification, and is easy to operate and grasp.

Description

technical field [0001] The invention relates to a polymerase chain reaction (PCR) amplification method in the field of biotechnology, in particular to a method for improving the polymerase chain reaction (PCR) amplification effect by utilizing uridine. Background technique [0002] Polymerase chain reaction (PCR), also known as in vitro enzymatic gene amplification, is a gene amplification technology that simulates DNA replication in vitro. This technology was invented by K.Mullis in 1985. Under the action of enzymes and the guidance of specific primers, a large number of gene duplications that only occur when organisms cell division and proliferation can be realized in a test tube in a very short period of time. Generally speaking, within a few hours. Amplify the gene of interest to a million-fold. [0003] Over the past 20 years, PCR technology has gradually matured through continuous progress and development, forming a complete set of technical systems. Conventional PCR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张治洲郑嘉祺徐仲吕志伟王有志
Owner 山东大正医疗器械股份有限公司
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