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Primers and method for detecting 79th site, 208th site and 435th site of CDA gene

A technology of CDA435-R and CDA435-F, which is applied in the fields of life science and biology, can solve the problems of affecting the blood concentration of gemcitabine, affecting adverse reactions and even curative effects, adverse reactions, etc., and achieves detection cost savings, low cost, and high specificity and accuracy effects

Inactive Publication Date: 2015-10-14
南昌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

However, the therapeutic window of this drug is small, and it is easy to cause adverse reactions mainly caused by myelosuppression.
Studies have shown that the single nucleotide polymorphism in the gene sequence encoding cytidine deaminase CDA may affect the blood concentration of gemcitabine, and then affect the adverse reactions and even the curative effect of gemcitabine.

Method used

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  • Primers and method for detecting 79th site, 208th site and 435th site of CDA gene
  • Primers and method for detecting 79th site, 208th site and 435th site of CDA gene
  • Primers and method for detecting 79th site, 208th site and 435th site of CDA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]The primers for detection of the 79th, 208th and 435th positions of the CDA gene include: forward and reverse primers for amplifying and covering the detection of the 79th, 208th and 435th positions of the CDA gene; the forward and reverse primers for the detection of the 79th position are respectively For CDA79-F and CDA79-R, the primers for determining the 208th position were CDA208-F and CDA208-R, respectively, and the primers for determining the 435th position were CDA435-F and CDA435-R. The base sequence of the extended primer specifically includes:

[0048] CDA79-F

TGTAAAACGACGGCCAGTAGAGTGTGAAGCACACGTAGG

CDA79-R

TGTAAAACGACGGCCAGTCTGCGCCTCTTCCTGTACATC

CDA208-F

TGTAAAACGACGGCCAGTCTCTGCTCTCTTCTCAGGGT

CDA208-R

TGTAAAACGACGGCCAGTGTTGGCTAAAGAGATGAATG

CDA435-F

TGTAAAACGACGGCCAGTTCTCTCACGCCAGCTTTGCC

CDA435-R

TGTAAAACGACGGCCAGTCTCAGGCTGGAGTGTAATCTG

[0049] The primers also include forward and rever...

Embodiment 2

[0062] The operation process of the blood DNA extraction kit (Tiangen Biology):

[0063] (1) Genomic DNA extracted from blood:

[0064] 1) Take 500uL of blood and add 1000uL 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 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0065] 2) Add 20 μl proteinase K solution and mix well.

[0066] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0067] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0068] 5) Put the solution and flocculent prec...

Embodiment 3

[0094] Four samples of clinical breast cancer patients were taken, and whether there was a CDA gene mutation in the four samples was detected. The genome was extracted, reagents were prepared and tested according to the method described in Example 2. Add 2 μl of each sample to the detection system PCR reaction solution. At the same time, make positive, negative, and blank controls. Detect with common PCR instrument, the time is 160 minutes.

[0095] sample number

mutation

1

no mutation

2

A79C

3

no mutation

4

no mutation

[0096] The forward sequencing result of the 79th position of the sample 1 sequence is shown in Figure 1, which is wild type. The forward sequencing results of the 79th position of the sample 2 sequence are as follows: figure 2 Shown is a heterozygous mutant.

[0097] The 208 and 435 sites of sample 1, sample 2, sample 3 and sample 4 were sequenced and compared, no mutation was found, and th...

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Abstract

The invention discloses primers and method for detecting the 79th site, 208th site and 435th site of a CDA gene. The primers comprise a forward primer, a reverse primer and a sequencing primer used for amplification of the 79th site, 208th site and 435th site of the CDA gene. The primers and the method disclosed by the invention are used for quickly detecting Lys27Gln, Ala70Thr and Thr145Thr of the CDA gene and provide a reference for a patient about using of difluorine nuclear anti-metabolism anti-tumor medicine gemcitabine.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to primers and methods for detecting the 79th, 208th and 435th positions of CDA gene. Background technique [0002] Gemcitabine is a difluoronuclear anti-metabolite antineoplastic drug that destroys cell replication, and it is widely used clinically in the first-line treatment of non-small cell lung cancer. Many other solid tumors are active. However, the therapeutic window of the drug is small, and it is easy to cause adverse reactions mainly caused by myelosuppression. Studies have shown that single nucleotide polymorphisms in the gene sequence encoding cytidine deaminase CDA may affect the blood concentration of gemcitabine, and then affect its adverse reactions and even curative effect after administration. Studies have shown that the CAD gene polymorphism sites include the mutation A79C at the 79th position (ie Lys27Gln), the mutation G208A at the 208th position ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 孙翠莲黎洪奋王淑一
Owner 南昌艾迪康医学检验实验室有限公司
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