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Folic acid heredity metabolism ability detection using mass spectrum

A technology of metabolic ability and folic acid, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of not fully disclosing the detection technology, staying in tumor detection, unthinkable, etc., to overcome the SNP position The effect of too few points, high degree of automation, and fast speed

Active Publication Date: 2015-09-30
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, based on the teachings of the above technologies, it is easy for researchers to think of detecting the above three sites of MTHFR by mass spectrometry to determine the best drug regimen for tumor patients, but it is difficult to think of adjusting and changing the three sites, and it is difficult to think of using It is used to guide folic acid users to take folic acid reasonably
[0025] In summary, the current technical problems are: the lack of methods and products that can simultaneously detect multiple folic acid-related polymorphic sites at one time, common detection technologies, such as sequencing, real-time fluorescent quantitative PCR, etc., all require site alignment Detect one by one, when there are many sites, the operation is complicated and the cost is high
However, the existing mass spectrometry detection technology has not fully disclosed the detection technology for the above multiple sites at the same time, and these technologies still remain in the relevant aspects of tumor detection

Method used

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  • Folic acid heredity metabolism ability detection using mass spectrum
  • Folic acid heredity metabolism ability detection using mass spectrum
  • Folic acid heredity metabolism ability detection using mass spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Primer design and synthesis.

[0105] For the folic acid-related SNPs are: MTHFR gene rs1801131 site (A1298C), MTHFR gene rs1801133 site (C677T) and MTRR gene rs1801394 site (A66G), design corresponding specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 6) and specific extension primer core sequences (SEQ ID No: 7 to SEQ ID No: 9).

[0106] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 6) at the 5' end of each PCR primer , common such as 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primer, thereby exceeding the detection window of the mass spectrometer.

[0107] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0108] The following detection process is performed with reference to the "Folic Acid Genetic ...

Embodiment 2

[0109] Example 2: DNA extraction from blood sheet.

[0110] The blood sheet used is a circular filter paper with a diameter of 2 cm. Take 50ul of whole blood, drop it on the center of the circular filter paper, and then dry it for later use. The specific steps of blood sheet DNA extraction are as follows:

[0111] (1) Prepare the scissors and pre-treat them with 75% alcohol. Then cut out the circular piece of paper with blood spots and cut it into very small pieces, put it into a 1.5ml centrifuge tube, add 20ul proteinase K, 20ulDTT and 500ul Lysis Buffer 1, mix well (vortex), and put in Place at 56°C for 2h (during this period, mix by inverting 3-4 times).

[0112] (2) Take out the centrifuge tube, centrifuge briefly, and use a pipette to transfer the supernatant as completely as possible to a new 1.5ml centrifuge tube to remove the interference of the filter paper on the next step of the experiment.

[0113] (3) Add 850ul Binding Buffer 2 and 150ul magnetic beads to the s...

Embodiment 3

[0122] Embodiment three: Biological experiment.

[0123] Using ABI 9700 PCR instrument, according to the instructions, the three polymorphic sites related to folic acid genetic metabolism genes were tested.

[0124] The components used in the kit for PCR, PCR product purification and single base extension are shown in Table 5:

[0125] table 5

[0126] serial number

component name

main ingredient

Specification

1

PCR mix

dNTPs, MgCl2, PCR primers

360ul / tube x 1 tube

2

PCR enzyme

Taq enzyme

24ul / tube x 1 tube

3

SAP Enzyme Mix

SAP enzyme

24ul / tube x 1 tube

4

Extension Primer Mix

extension primer

24ul / tube x 1 tube

5

elongase mix

iPLEX enzymes, ddNTPs

24ul / tube x 1 tube

6

positive control

Human Genomic DNA (30ng / ul)

40ul / tube x1 tube

[0127] According to the manual, the specific operation method is as follows:

[0128] 1. PCR amplificati...

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Abstract

The invention relates to folic acid heredity metabolism ability detection using mass spectrum, and discloses a primer system for detecting folic acid heredity metabolism ability-associated gene polymorphism sites (SNP). A product produced on the basis of the primer system can realize simultaneous detection of the folic acid heredity metabolism ability-associated gene polymorphism sites. The product is used to detect the folic acid heredity metabolism ability-associated gene polymorphism sites in order to provide reference basis for guidance of reasonable administration of women preparing for pregnancy and pregnant women. The system and the product allow three gene polymorphism sites on different genes to be simultaneously detected in a reaction system; and compared with sequencing, real-time fluorescent quantitative PCR and other technologies, a method adopting the system or the product has the advantages of low cost, simple operation, and increased accuracy and sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining the polymorphic site (SNP) of a gene related to folic acid genetic metabolism ability, specifically using multiplex PCR technology, single base extension technology and mass spectrometry technology , a method for detecting three genetic polymorphic sites of folic acid genetic metabolism ability and a corresponding kit. Background technique [0002] Folic acid is one of the B vitamins. It participates in the synthesis of DNA, RNA, protein and other important compounds. It plays an important role in the body's metabolic process. It is often used in nutritional supplements during pregnancy to prevent diseases such as neural tube defects. [0003] The top 5 birth defects in my country include: congenital heart disease, neural tube defects, cleft lip and palate, Down syndrome, and polydactyly (toes). According to the statistical results in 200...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2535/125C12Q2565/627
Inventor 马庆伟张海燕钟逾李学媛林燕向华
Owner BIOYONG TECH
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