WO2014032475A1 - Primers, kit and method for identifying polymorphism of human mthfr gene c677t - Google Patents

Description

 Primer, kit and method for identifying MTHFR gene C677T polymorphism  Technical field

 The present application relates to primers, kits and methods for identifying the human MTHFR gene C677T polymorphism.

Background technique

The most common mutation in the MTHFR gene (Accession No. NM_005957) is a mutation at position 677 to C, which affects the activity of the MTHFR enzyme. The MTHFR enzyme is one of the important enzymes in the metabolism of homocysteine (Hcy), and the decrease in MTHFR enzyme activity leads to an increase in the concentration of Hcy in the body. Hcy is a vascular-damaging amino acid whose elevated concentration can lead to vascular endothelial damage leading to atherosclerosis. The study found that the polymorphism of MTHFR gene C677T is related to serum Hcy and folic acid levels in patients with cerebrovascular disease.

Summary of the invention

The present application provides a primer, a kit and a method capable of identifying a C677T polymorphism of the MTHFR gene.

The present application provides a primer for identifying a human MTHFR gene C677T polymorphism, comprising an upstream primer and a downstream primer, the sequence of the upstream primer being SEQ ID NO: The sequence shown in 1 and/or the sequence shown in SEQ ID NO: 2, and the sequence of the downstream primer is the sequence shown in SEQ ID NO: 3.

Further, the sequence of the upstream primer is the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2.

A kit for identifying a human MTHFR gene C677T polymorphism, comprising a first pair of primers and a second pair of primers, wherein the sequence of the upstream primer of the first pair of primers is SEQ ID As shown by NO: 1, the sequence of the upstream primer of the second pair of primers is shown in SEQ ID NO: 2, and the sequence of the downstream primers of the first and second pairs of primers are all shown in SEQ ID NO: 3.

A method for identifying a human MTHFR gene C677T polymorphism using the primers described.

Further, for the sequences, respectively, SEQ ID NO: 1, SEQ ID The upstream and downstream primers shown by NO: 3, the sequence of the amplified product is shown in SEQ ID NO: 4; for the sequences, respectively, SEQ ID NO: 2, SEQ ID The upstream and downstream primers shown by NO: 3 have the sequence of the amplified product as shown in SEQ ID NO: 5.

A method for identifying a human MTHFR gene C677T polymorphism, comprising the following steps:

 a) obtaining the genomic DNA of the human to be tested;

b) using the first pair of primers and the second pair of primers, respectively, using the genomic DNA of the human to be tested as a template, performing a PCR amplification reaction to obtain first and second amplification products respectively; wherein, the first pair of primers The sequence of the upstream primer is SEQ ID NO: 1, the sequence of the downstream primer of the first pair of primers is shown in SEQ ID NO: 3; the sequence of the upstream primer of the second pair of primers is SEQ ID NO: 2, the sequence of the downstream primer of the second pair of primers is shown in SEQ ID NO: 3;

c) performing electrophoresis on the first and second amplification products, respectively, to obtain a polymorphism identification result.

Further, the sequence of the first amplification product is as shown in SEQ ID NO: 4, and the sequence of the second amplification product is as SEQ ID NO: 5 is shown.

The beneficial effects of the present application are as follows: 1) By designing a specific primer, it is possible to identify whether the MTHFR gene 677 site is mutated, thereby distinguishing whether it is wild-type, heterozygous or mutant. 2) Direct electrophoresis after PCR amplification, which simplifies the identification step, makes the operation simpler, and reduces the cost.

DRAWINGS

Fig. 1 is a graph showing the results of electrophoresis of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

The invention uses the ARMS-PCR technology to identify the C677T polymorphism of the MTHFR gene in the patient genome, and directly electrophoresis after the PCR reaction can distinguish the wild type (CC type), the heterozygous type (CT type) and the mutant type (TT). type).

Two pairs of primers were designed according to the MTHFR gene sequence. Both pairs of primers can be used to amplify the sequence near the 677 site of the MTHFR gene. The two pairs of primers are defined as a first pair of primers and a second pair of primers, respectively.

The sequence of the upstream primer of the first pair of primers is: GAAGGTGTCTGCGGGCGC (SEQ ID NO: 1), this sequence was used to identify the sequence of the MTHFR gene at position 677 (ie, the 677 site was not mutated).

The sequence of the upstream primer of the second pair of primers is: AGAAGGTGTCTGCGGGCGT (SEQ ID NO: 2), this sequence was used to identify the sequence of the MTHFR gene at position 677 (ie, the 677 site was mutated from C to T).

The sequences of the downstream primers of the first and second pairs of primers are: TGGGAAAGATCCCGGGGACG (SEQ ID NO: 3).

In each of the above sequences, the last base of the two upstream primer sequences is the template to be tested.

Paired bases; the third base of the last upstream primer sequence is an additional mismatch base, with the goal of further increasing the specificity of the identity.

 The method for identifying the M677R gene C677T polymorphism of the present invention comprises the following steps:

1) Extract DNA

Take 50 microliters of whole blood from the patient and use the kit of Qiagen to extract the patient's genomic DNA.

2) Using the first pair of primers and the second pair of primers, respectively, using the patient genomic DNA as a template to be tested, performing a PCR amplification reaction to obtain a PCR amplification product.

The reaction system during PCR amplification is: premix (Takara PCR Master Mix) 10 μL, 1 μL of template to be tested, 0.4 μL of each pair of primers (10 μM), and 8.60 μL of pure water, that is, the total volume of the PCR reaction solution was 20 μL.

The reaction conditions for PCR amplification were: 95 ° C for 3 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C 45 Seconds, 35 cycles, saved at 4 °C.

The sequence of the amplified product obtained using the first pair of primers is:

GAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCA (SEQ ID NO: 4).

The sequence of the amplified product obtained using the second pair of primers is:

GAGAAGGTGTCTGCGGGAGTCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCA (SEQ ID NO: 5).

3) 2% agarose electrophoresis was performed on the amplification products of the first pair of primers and the amplification products of the second pair of primers respectively. The electrophoresis results are shown in Figure 1. The MTHFR gene C677T polymorphism was identified, and the electrophoresis results and utilization were used. Prior art PCR amplification followed by cleavage and final electrophoresis results were consistent.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. A number of simple derivations or substitutions may be made by those skilled in the art without departing from the inventive concept.

Claims (7)

 A primer for identifying a human MTHFR gene C677T polymorphism, comprising an upstream primer and a downstream primer, the sequence of the upstream primer being SEQ ID The sequence shown by NO: 1 and/or the sequence shown by SEQ ID NO: 2, and the sequence of the downstream primer is the sequence shown by SEQ ID NO: 3. The primer for identifying a human MTHFR gene C677T polymorphism according to claim 1, wherein the sequence of the upstream primer is SEQ ID NO: the sequence shown by 1 and the sequence shown by SEQ ID NO: 2. A kit for identifying a human MTHFR gene C677T polymorphism, comprising: a first pair of primers and a second pair of primers, wherein the sequence of the upstream primer of the first pair of primers is SEQ ID As shown by NO: 1, the sequence of the upstream primer of the second pair of primers is as shown in SEQ ID NO: 2, and the sequence of the downstream primers of the first and second pair of primers is as SEQ ID. NO: 3 is shown.  A method for identifying a human MTHFR gene C677T polymorphism, which comprises using the primer of claim 2.  A method for identifying a human MTHFR gene C677T polymorphism according to claim 4, wherein the sequences are SEQ ID NO: 1, SEQ, respectively. The upstream and downstream primers shown by ID NO: 3, the sequence of the amplified product is shown in SEQ ID NO: 4; for the sequence, respectively, SEQ ID NO: 2, SEQ ID The upstream and downstream primers shown by NO: 3 have the sequence of the amplified product as shown in SEQ ID NO: 5. A method for identifying a human MTHFR gene C677T polymorphism, comprising the steps of:  a) obtaining the genomic DNA of the human to be tested; b) using the first pair of primers and the second pair of primers, respectively, using the genomic DNA of the human to be tested as a template, performing a PCR amplification reaction to obtain first and second amplification products respectively; wherein, the first pair of primers The sequence of the upstream primer is SEQ ID NO: 1, the sequence of the downstream primer of the first pair of primers is shown in SEQ ID NO: 3; the sequence of the upstream primer of the second pair of primers is SEQ ID NO: 2, the sequence of the downstream primer of the second pair of primers is shown in SEQ ID NO: 3; c) performing electrophoresis on the first and second amplification products, respectively, to obtain a polymorphism identification result. The method for identifying a human MTHFR gene C677T polymorphism according to claim 6, Characterized in that the sequence of the first amplification product is as shown in SEQ ID NO: 4, and the sequence of the second amplification product is as SEQ ID. NO: 5 is shown.

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