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Pair of specific primers and probe for detection of MTHFR gene chip

A technology of specificity and primer pairs, applied in the field of molecular biology, can solve the problems of cumbersome operation, long detection period, and increased risk of giving birth to cleft lip and palate babies, and achieve good signal-to-noise ratio, good specificity, and avoid uncertain factors Effect

Inactive Publication Date: 2014-01-22
SHANGHAI BAIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have confirmed that folic acid metabolism disorders caused by defects in folic acid metabolism-related genes lead to DNA hypomethylation and non-disjunction of chromosomes, which is a risk factor for Down syndrome (DS)
Other studies have found that mothers who carry the 677TT genotype and have low intakes of folic acid supplements or dietary folic acid have a significantly increased risk of having babies with cleft lip and palate
At present, methods for determining MTHFR genotypes mainly use methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), manual or automatic sequencing, and PCR with sequence-specific primers. Difficult to be accurate and difficult to meet the requirements of clinical testing

Method used

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  • Pair of specific primers and probe for detection of MTHFR gene chip
  • Pair of specific primers and probe for detection of MTHFR gene chip
  • Pair of specific primers and probe for detection of MTHFR gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1 gene chip

[0055] Aldehyde-modified glass slides (product number: BSM03011, Shanghai Bio Technology Co., Ltd.). The following probes were artificially synthesized (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), dissolved in water to a concentration of 100pmol / ul, and then mixed in equal proportions with 2× spotting buffer (product number: BST02010, Shanghai Bio-Technology Co., Ltd.) . Then, use the GSM417 sample spotting instrument of Affymetrix Company to make points such as figure 1 array of . Leave overnight at room temperature.

[0056] Each specific probe sequence is as follows:

[0057] The specific oligonucleotide probe for detection site 677C is, NH 2 - TTTTTTTTTTTTTTTT TGCGGGAGCCGATTTCAT; The specific oligonucleotide probe for detection site 677T is, NH 2 -TTTTTTTTTTTTTTTTGTCTGCGGGAGTCGATTTCA-3'. That is, each of the above-mentioned probes is SEQ ID No.1 and 4 in the sequence table, and the 5' end also cont...

Embodiment 2

[0059] Example 2 Preparation of chromosomal DNA

[0060] Use the blood DNA extraction kit from Shanghai Bio-Tech Co., Ltd. and follow the instructions as follows:

[0061] Adsorption column activation:

[0062] Put the adsorption column in the collection tube, add 500 μL buffer BH1, let it stand for 2-3 minutes, and centrifuge at 12,000 rpm (9,500×g) for 30 s; discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 500 μL of buffer BH2 ​​to the adsorption column, centrifuge at 12,000 rpm (9,500×g) for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube for use.

[0063] Operating procedure:

[0064] (1) Add 20 μL of proteinase K to the bottom of a 1.5 mL centrifuge tube with a pipette.

[0065] (2) Add 200 μL of blood sample into the centrifuge tube.

[0066] (3) Add 200 μL buffer BL to the centrifuge tube, shake and mix for 15 seconds.

[0067] (4) Inc...

Embodiment 3

[0076] Embodiment 3 amplifies the MTHFR gene fragment by PCR method with the primer provided by the present invention

[0077] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize primers, and the primer information is as follows.

[0078] Upstream SEQ ID No.7: 5'-GTTGGAAGGTGCAAGATCAG-3',

[0079] Downstream SEQ ID No.8: 5'-CTCACGACTCAAGCGACTCA-3',

[0080] Also, the 5' end of the primer was modified with biotin.

[0081] Then dissolve and dilute to 10 pmol / μl with water. Prepare the PCR amplification system with the purchased Taq enzyme (TaKaRa), 10× buffer (TaKaRa), dNTP (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), pure water, and the PCR amplification template obtained in Example 2 according to the following formula :

[0082] Table 1

[0083]

[0084] Use a PCR instrument (TC-96 / G / H(b) PCR amplification instrument, Hangzhou Bioer) to amplify according to the following procedures: 50°C for 5min, 94°C for 5min, then 94°C...

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Abstract

The invention relates to the molecular biology field, and discloses a specific oligonucleotide probe for detection of an SNP locus of rs1801133677C / T of an MTHFR gene and a pair of specific primers for amplification of a target zone containing the target locus to be detected during detection of the above SNP locus. The oligonucleotide probe can hybridize with different genotypes of the 677th locus of the MTHFR gene specifically. The pair of specific primers can amplify a target zone containing the target locus to be detected. By utilization of a chip hybridization method, the probe and the above amplified zone are hybridized for detection of the MTHFR genotypes, which provides references for evaluation of suspicious patients with thrombophilia, increase of homocysteine level and change of folic acid metabolism, and provides information for doctors for correct selection of medicines and reasonable adjustment of medicine dosage.

Description

technical field [0001] The invention relates to the fields of molecular biology and nucleic acid detection, specifically designing specific primer pairs for amplifying MTHFR genes and specific probes for MTHFR gene chip detection. Background technique [0002] Cerebrovascular disease seriously endangers human health with its high morbidity, high mortality, and high disability rate, and is currently one of the most common causes of death for humans. In recent years, hyperhomocysteinemia as a new stroke risk factor has gradually attracted people's attention. Current research has confirmed that hyperhomocysteinemia is an independent risk factor for stroke, and about 20% to 50% of stroke patients have different degrees of homocysteinemia. [0003] Methylenetetrahydrofolate-reductase (MTHFR) is a key enzyme in the metabolic pathway of homocysteine ​​(Hcy) in the human body. Insufficient content or decreased activity will directly lead to the loss of homocysteine ​​in the body. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 朱滨邢军芬孙悦张宇张佳琦张艳张莹
Owner SHANGHAI BAIO TECH
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