Method for detecting polymorphism of folate metabolism related genes through whole-blood direct nucleic acid amplification

A technology for gene polymorphism and nucleic acid amplification, which is applied in the field of primers for direct amplification of nucleic acid in whole blood to detect folic acid metabolism-related gene polymorphisms, can solve the problems of increasing the workload of testing personnel, testing costs, and sample contamination, and achieves Conducive to review, 100% accuracy, simple operation

Inactive Publication Date: 2017-06-20
浙江中迪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 3. Genomic DNA needs to be extracted, which will increase the workload

Method used

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  • Method for detecting polymorphism of folate metabolism related genes through whole-blood direct nucleic acid amplification
  • Method for detecting polymorphism of folate metabolism related genes through whole-blood direct nucleic acid amplification
  • Method for detecting polymorphism of folate metabolism related genes through whole-blood direct nucleic acid amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] PCR of blood samples from different anticoagulant sources: Collect fresh blood from the same person with different anticoagulants, and collect them in EDTA anticoagulant tubes, citrate anticoagulant tubes and heparin anticoagulant tubes respectively.

[0078] Prepare the PCR reaction solution as follows,

[0079] components Volume μl 10×BR Taq Buffer 3 dNTP 2.4 Forward and reverse primer mix 20μM 2 ddH20 19.4 blood sample 3 BR Taq 0.2

[0080] The PCR reaction conditions are as follows:

[0081] 98°C for 3 minutes;

[0082] 98°C for 30s, 58°C for 30s, 72°C for 30s, 40cycle;

[0083] 72℃10min,

[0084] 4℃hold.

[0085] The PCR electrophoresis images of blood from different anticoagulation sources are shown in figure 2 ,in

[0086] M: 100bp DNA ladder, from top to bottom are 1500bp, 1200bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, of which 500bp is a bright band;

[0087] 1: MTHFR-C677T of heparin...

Embodiment 2

[0095] Detect 7 human blood samples from different sources, and prepare the PCR reaction solution according to the following method,

[0096] components Volume μl 10×BR Taq Buffer 3 dNTP 2.4 Forward and reverse primer mix 20μM 2 ddH20 19.4 blood sample 3 BR Taq 0.2

[0097] The PCR reaction conditions are as follows:

[0098] 98°C for 3 minutes;

[0099] 98°C 30s, 58°C 30s, 72°C 30s, 40 cycles;

[0100] 10min at 72°C,

[0101] 4°C hold.

[0102] The PCR electrophoresis graphs of 7 human blood samples from different sources are shown in image 3 ,in

[0103] M: 100bp DNA ladder, from top to bottom are 1500bp, 1200bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp. Among them, 500bp is a bright band.

[0104] 1: MTHFRC677T site of citrate anticoagulated blood sample 1

[0105] 2: MTHFRC677T site of citrate anticoagulated blood sample 2

[0106] 3: MTHFRC677T site of citrate anticoagulated blood sample 3...

Embodiment 3

[0120] The blood of the same sample was tested for 5 times, and the PCR reaction solution was prepared according to the following method,

[0121] components Volume μl 10×BR Taq Buffer 3 dNTP 2.4 Forward and reverse primer mix 20μM 2 ddH20 19.4 blood sample 3 BR Taq 0.2

[0122] The PCR reaction conditions are as follows:

[0123] 98°C for 3 minutes;

[0124] 98°C 30s, 58°C 30s, 72°C 30s, 40cycle;

[0125] 10min at 72°C,

[0126] 4°C hold.

[0127] The PCR electrophoresis figure of the blood of this sample is repeated 5 times to see Figure 4 ,in

[0128] M: 100bp DNA ladder, from top to bottom are 1500bp, 1200bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp. Among them, 500bp is a bright band.

[0129] 1-5: The blood of this sample was tested for MTHFRC677T site repeated 5 times.

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Abstract

The invention discloses a method for detecting polymorphism of folate metabolism related genes through whole-blood direct nucleic acid amplification. The method comprises the following steps: adopting a whole-blood sample as a starting material, adding primers for amplifying MTHFR C677T and MTHFR A1298C polymorphic region fragments, as well as blood resistance taq polymerase buffer and blood resistance taq polymerase in a nucleic acid amplification reaction reagent, so that a PCR system is constituted for directly conducting a nucleic acid amplification reaction; and a PCR product is directly delivered to a commercial company for being subjected to sequencing, so that workload is reduced. A result can be clearly judged by analyzing a mutation site on a sequencing diagram, with an accuracy rate reaching 100%. The method is simple to operate, low in cost, rapid and accurate, and high in throughput; detection of normal hospitals in second and third-tier cities can be satisfied; and the method is applicable to clinical detection and popularization.

Description

technical field [0001] The invention relates to molecular biology detection, in particular to a primer, a primer set, a kit and a detection method for directly amplifying nucleic acid from whole blood to detect polymorphisms of genes related to folic acid metabolism. Background technique [0002] PCR is a modern technology that uses DNA polymerase to amplify a very small sample by tens of billions or even hundreds of billions of times, and its starting template is usually genomic DNA. Human genomic DNA is mainly derived from blood and needs to be extracted. In addition to commercial kits, there are many other extraction methods, such as: phenol / chloroform extraction method, potassium iodide method, direct boiling method, high-salt precipitation method, etc. . Although these methods are effective, the operation steps are cumbersome and easy to cause pollution, and basically all need to use devices such as constant temperature water baths, low-temperature high-speed centrifug...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6806C12Q1/6883C12Q2600/156
Inventor 苏宇曲孙涛
Owner 浙江中迪生物科技有限公司
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