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Quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity

A specific and sensitive technology, applied in the field of quantitative PCR, to achieve the effect of good specificity, convenient use and good versatility

Active Publication Date: 2013-11-27
苏州依科赛生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Therefore, need a kind of quantitative PCR method with high sensitivity and good specificity, about the quantitative PCR method of the present invention, there is no report yet

Method used

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  • Quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity
  • Quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity
  • Quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] One, the reagent of the quantitative PCR reaction system of the present embodiment is as follows:

[0026] 10×Buffer (Mg 2+ free);

[0027] 10×Gelgreen I;

[0028] 10×PCR protectant (fetal bovine serum albumin);

[0029] Each 10mmol / L solution of dATP, dUTP, dGTP and dCTP;

[0030] 25mmol / L Mg 2+ ;

[0031] 5U / μL hTaq enzyme;

[0032] Sterile double distilled water.

[0033] Two, the application of the quantitative PCR method of this embodiment

[0034] (1) Preparation of DNA to be tested: DNA was extracted from mouse blood according to conventional methods, and EGF gene was amplified.

[0035] (2) Synthesis of primers.

[0036] Forward primer: TATAGATATCATGAATAGTTATCCAGGATGCCCA

[0037] Reverse primer: TATA GAATTCTCAACGCAGTTCCCCACCATCGTAG.

[0038] (3) The establishment of the PCR reaction system, the total reaction volume is 25 μL, and the reagents are added according to the table below.

[0039] Table 1 PCR reaction system

[0040] .

[0041] (4) qPC...

Embodiment 2

[0050] The reagents of the quantitative PCR reaction system of the present embodiment are as follows:

[0051] 10×Buffer (Mg 2+ free);

[0052] 10×Gelgreen I;

[0053] 10×PCR protectant (Tween 20);

[0054] Each 5mmol / L solution of dATP, dUTP, dGTP and dCTP;

[0055] 20mmol / L Mg 2+ ;

[0056] 5U / μL hTaq enzyme;

[0057] 10 μmol / L forward primer;

[0058] 10μmol / L reverse primer;

[0059] Sterile double distilled water.

[0060] The working concentration of the fluorescent dye Gelgreen I is 0.5ט1×. The working concentration of the hot start hTaq enzyme is 0.01-0.1 U / μL. The working concentration of the dATP solution, dUTP solution, dGTP solution or dCTP solution is 100-200 μmol / L.

[0061] Prepare the DNA to be tested according to the conventional method, establish the PCR reaction system, and carry out the qPCR reaction.

Embodiment 3

[0063] The reagents of the quantitative PCR reaction system of the present embodiment are as follows:

[0064] 10×Buffer (Mg 2+ free);

[0065] 10×Gelgreen I;

[0066] 10×PCR protectant (dithiothreitol);

[0067] Each 10mmol / L solution of dATP, dUTP, dGTP and dCTP;

[0068] 30mmol / L Mg 2+ ;

[0069] 5U / μL hTaq enzyme;

[0070] 10 μmol / L forward primer;

[0071] 10μmol / L reverse primer;

[0072] Sterile double distilled water.

[0073] The working concentration of the fluorescent dye Gelgreen I is 0.5ט1×. The working concentration of the hot start hTaq enzyme is 0.01-0.1 U / μL. The working concentration of the dATP solution, dUTP solution, dGTP solution or dCTP solution is 100-200 μmol / L.

[0074] Prepare the DNA to be tested according to the conventional method, establish the PCR reaction system, and carry out the qPCR reaction.

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Abstract

The invention discloses a quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity. Fluorescent dye GelgreenI and hot start hTaq enzyme are added into a PCR system, and the fluorescent dye GelgreenI can be specifically combined with DNA double chains to send out a fluorescent signal. The high-sensitivity and high-specificity quantitative PCR method provided by the invention adopts the hot start hTaq enzyme and proper magnesium ion concentration, so non-specific amplification can be inhibited well, false positive and high background results are avoided, and a solubility curve can obtain single peak; the fluorescent dye GelgreenI is low in price, convenient to use, good in universality, high in sensitivity and applicable to a qPCR technology; the invention provides a new quantitative PCR method for molecular biology research.

Description

technical field [0001] The invention relates to the technical field of quantitative PCR, in particular to a quantitative PCR method with improved sensitivity and specificity. Background technique [0002] Real-time quantitative PCR (qPCR) is a technique for quantitative and qualitative analysis of the starting template by detecting the fluorescence signal corresponding to each cycle of PCR amplification product in real time during the PCR amplification process. The main difference between qPCR technology and ordinary PCR technology is that ordinary PCR involves more manual participation, and the data may not be particularly accurate. It has the advantages of short detection time, simple operation, high specificity, good repeatability, high sensitivity, high resolution, wide detection range, and accurate quantification. Due to its many advantages, qPCR technology has now been applied to various fields of life science research, such as differential expression analysis of gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 蒋国成陈旭陈刚吴小祝
Owner 苏州依科赛生物科技股份有限公司
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