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Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5)

A cytochrome, genotyping technology, used in DNA/RNA fragmentation, recombinant DNA technology, etc.

Inactive Publication Date: 2013-10-23
天津市秀鹏生物技术开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Today, the detection of CYP3A5 genotyping mostly uses sequencing and PCR-RFLP methods, but both methods have certain limitations

Method used

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  • Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5)
  • Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5)
  • Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] According to the CYP3A5 allele sequence released by the National Center for Biotechnology Information (NCBI Gene Bank), a primer set for detecting CYP3A5 genotyping was designed, which includes CY3A5*1 primer pair, CY3A5*2 primer pair , CY3A5*3 primer pair, CY3A5*4 primer pair, CY3A5*5 primer pair, CY3A5*6 primer pair, and internal reference primer pair.

Embodiment 2

[0031] The preparation method of the test kit for detecting CYP3A5 genotyping is:

[0032] (1) According to the well location map of CYP3A5 gene-specific primers (see figure 1 ), respectively coated CY3A5*1 primer pair, CY3A5*2 primer pair, CY3A5*3 primer pair, CY3A5*4 primer pair, CY3A5*5 primer pair, CY3A5*6 primer pair and internal reference primer pair to the corresponding primer plate position, drying process;

[0033] (2) Prepare 440 μl concentrated dNTP-Buffer according to the combination of 220mM dNTP, 3.5mM Mg2+, 500mM KCL, 100mM Tris-HCL, and 1% TritonX-100;

[0034] (3) The primer plate coated with primer pairs includes 10 wells of specific primer wells per person, and the dNTP-Buffer is concentrated to form a kit for human CYP3A5 genotyping detection.

Embodiment 3

[0036] Use the operation process

[0037] 1. Preparation of 2.5% agarose gel:

[0038] 1. Add 2.5g of agarose to 100ml of 1×TBE solution (Tris-borate-EDTA solution) and heat until a uniform gel solution is formed. Then add an appropriate amount of electrophoresis dye and mix well.

[0039] 2. Put the gel tank in a horizontal position, add an appropriate amount of gel solution into the gel tank, and insert the electrophoresis comb. Move the gel tank horizontally back and forth to ensure even coverage of the gel solution.

[0040]3. After the gel is completely solidified, pull out the electrophoresis comb vertically.

[0041] 2. PCR cycle parameters are shown in Table 1:

[0042] 1

96℃ / 2min

1cycle

2

96°C / 20sec, 68°C / 60sec

5cycles

3

96°C / 20sec, 65°C / 50sec, 72°C / 45sec

10cycles

4

96°C / 20sec, 63°C / 50sec, 72°C / 45sec

15cycles

5

72℃ / 5min

1cycle

6

Store at 4°C

[0043] Table 1 Ampli...

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Abstract

The invention discloses a primer group and a kit for detecting genetic typing of CYP3A5. The primer group for detecting CYP3A5 gene typing comprises a CY3A5*1 primer pair, a CY3A5*2 primer pair, a CY3A5*3 primer pair, a CY3A5*4 primer pair, a CY3A5*5 primer pair, a CY3A5*6 primer pair and an internal reference primer pair. The application of the kit helps to realize good typing for CYP3A5 gene; and in clinical application of organ transplantation, good guidance effects on individualized use and secure use of immunosuppressant are realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a kit for detecting CYP3A5 genotyping. Background technique [0002] The CYP3A5 gene is located in the q21.1-22.1 region of human chromosome 7. The full length of the gene is 31.8 kb, including 13 exons, encoding 502 amino acids. The current distribution in the Chinese population is mainly CYP3A5*3. There is an A>G change at position 6986 of the CYP3A5*3 allele, which creates a cryptic acceptor splice site in the 3rd intron, which promotes the exon-like sequence within the gene (pseudo exon) into mature mRNA and includes subsequent deletion of exon (or) insertion of its gene sequence. These aberrant splicing resulted in several in-frame premature stop codons. The mRNA of the mutant CYP3A5 is more rapid and unstable than that of the wild type, and this mechanism becomes nonsense codon-mediated mRNA degradation, which promotes the degradation of PTC-containing mRNA, which is a pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵卫军
Owner 天津市秀鹏生物技术开发有限公司
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