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C829T single nucleotide polymorphism assay kit of DHFR

A single nucleotide polymorphism and detection kit technology, which is applied in the fields of life science and biology, can solve the problem of decreased function, and achieve the effects of low cost, guaranteed accuracy and sensitivity, and simple operation

Active Publication Date: 2013-01-16
北京艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although MTX can competitively inhibit DHFR, when the affinity between MTX and DHFR decreases, the effect of MTX on inhibiting the enzyme will decrease significantly

Method used

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  • C829T single nucleotide polymorphism assay kit of DHFR
  • C829T single nucleotide polymorphism assay kit of DHFR
  • C829T single nucleotide polymorphism assay kit of DHFR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] C829T single nucleotide polymorphism detection kit for detecting DHFR, including erythrocyte lysate, DNA extraction solution, PCR reagent, single strand purification reagent and sequencing reagent, of which:

[0033] (i) PCR amplification reagent: 2*PCR Buffer, 10mM dNTP, 5U / ul Taq enzyme, amplification primers (SEQ NO.1, SEQ NO. 2) and sterilized water; the sequences of SEQ NO1 and SEQ NO 2 are :

[0034] SEQ NO. 1: ACTAAGTGCTTCTCCAAGACC,

[0035] SEQ NO.2: Biotin-AATGTCAAGGACTGGCAAGAG;

[0036] (ii) Reagent for single-strand purification: streptavidin bead, 70% (V / V) ethanol, denaturing solution, 1×Wash Buffer, binding buffer, annealing buffer;

[0037] (iii) Sequencing reagents: DNA polymerase, ATP sulfurylase, luciferase, apyrase, substrate APS, fluorescein and dNTP (dNTP is dATP, dTTP, dCTP, dGTP) and sequencing primers (SEQ NO. 3). The sequence of SEQ NO.3 is: AGTCCCCAGCACCTG.

[0038] The using method of above-mentioned test kit comprises the following steps...

Embodiment 2

[0046] The concrete usage method of kit of the present invention:

[0047] 1. DNA extraction

[0048] 1) Take 300uL of blood and add 900uL of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 10,000rpm for 1 minute (if the maximum speed of the centrifuge is not allowed, centrifuge at 3,000rpm for 5 minutes), absorb the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0049] 2) Add 20 μl proteinase K solution, mix well, add 200 μl buffer GB, mix well by inverting, place at 70°C for 10 minutes, the solution should become clear, briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0050] 3) Add 200 μl absolute ethanol, vortex and mix well for 15 seconds, at this time flocculent sediment may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0051] 4) Put the so...

Embodiment 3

[0065] Clinical Sample Testing

[0066] The clinical samples A, B, and C to be tested were taken, and the genome was extracted, reagents were prepared and tested according to the method described in Example 2.

[0067] After the PCR product is purified, it is sequenced, and the obtained sequence is compared with the standard sequence to judge the result.

[0068] The sequencing results of sample A are shown in the figure figure 1 As shown, the sequencing result is: CTA C AGTGAG, the sample is CC type (wild type), and the red box in the figure is the judgment area of ​​sample A genotype.

[0069] The sequencing results of sample B are shown in the figure figure 2 As shown, the sequencing result is: CTA C(T) AGTGAG, the sample is CT type (heterozygous type), and the red box in the figure is the judgment area of ​​the sample B genotype.

[0070] The sequencing results of sample C are shown in the figure image 3 As shown, the sequencing result is: CTA T AGTGAG, the...

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Abstract

The invention discloses a C829T single nucleotide polymorphism assay kit of DHFR (dihydrofolate reductase). The kit comprises a red blood cell lysate, a DNA extracting solution, a PCR (Polymerase Chain Reaction) reagent, a single-chain purification reagent and a sequencing reagent, and is characterized in that the PCR reagent comprises specific augmentation primers SEQNO.1 and SEQNO.2, whose sequences are respectively ACTAAGTGCTTCTCCAAGACC and Biotin-AATGTCAAGGACTGGCAAGAG; and the sequencing reagent comprises a specific sequencing primer SEQNO.3 whose sequence is AGTCCCCAGCACCTG.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a DHFR C829T single nucleotide polymorphism detection kit, which can detect the DHFR (C829T) polymorphism site by using gene sequencing technology. Background technique [0002] Myelosuppression restricts the application of various chemotherapeutic drugs, making it difficult to achieve the effect of curing or inhibiting tumor growth, and some drug resistance genes are the molecular basis of myelosuppression, such as dihydrofolate reductase gene (dihydrofolate reductase, DHFR). [0003] DHFR is a key enzyme in the metabolism of folic acid in cells, with a molecular weight of 22KD. With the participation of reduced coenzyme II (NADPH), DHFR can catalyze folic acid into dihydrofolate, and the latter transports carbon elements for the synthesis of thymidine and purine nucleosides. Therefore, DHFR is the synthesis of DNA, RNA and protein. one of the key enzymes. In addi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q1/6883C12Q2600/106C12Q2600/156C12Q2565/301C12Q2531/113
Inventor 陈奕磊汝昆徐建成王淑一孙翠莲
Owner 北京艾迪康医学检验实验室有限公司
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