Human creatine kinase: Isolation and sequence analysis of cDNA clones for the B subunit, development of subunit specific probes and determination of gene copy number

Abstract Background: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. Methods: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing™ technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. Results: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0–4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. Conclusions: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

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Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations

Drug Discoveries & Therapeutics ◽

10.5582/ddt.2017.01057 ◽

2017 ◽

Vol 11 (6) ◽

pp. 336-341 ◽

Cited By ~ 3

Author(s):

Yutaro Motoi ◽

Kazufumi Watanabe ◽

Hiroyuki Honma ◽

Yousuke Tadano ◽

Hiroshi Hashimoto ◽

...

Keyword(s):

Cytochrome P450 ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Copy Number Variations ◽

Gene Copy ◽

Cytochrome P450 2D6 ◽

Sulfotransferase 1A1 ◽

P450 2D6

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Application of a Modified Real-Time PCR Technique for Relative Gene Copy Number Quantification to the Determination of the Relationship between NKX3.1 Loss and MYC Gain in Prostate Cancer

Clinical Chemistry ◽

10.1373/clinchem.2004.045013 ◽

2005 ◽

Vol 51 (3) ◽

pp. 649-652 ◽

Cited By ~ 20

Author(s):

Roland Kindich ◽

Andrea R Florl ◽

Volker Jung ◽

Rainer Engers ◽

Mirko Müller ◽

...

Keyword(s):

Prostate Cancer ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

The Relationship ◽

Relative Gene

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Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR

Journal of Experimental Botany ◽

10.1093/jexbot/53.373.1515 ◽

2002 ◽

Vol 53 (373) ◽

pp. 1515-1520 ◽

Cited By ~ 9

Author(s):

M. Honda

Keyword(s):

Arabidopsis Thaliana ◽

Copy Number ◽

Transgenic Arabidopsis ◽

Gene Copy Number ◽

Gene Copy ◽

Competitive Pcr ◽

Transgenic Arabidopsis Thaliana

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Determination of Foreign Gene Copy Number in Stably Transfected Cell Lines by Southern Transfer Analysis

Gene Transfer and Expression Protocols ◽

10.1385/0-89603-178-0:381 ◽

2003 ◽

pp. 381-396 ◽

Cited By ~ 1

Author(s):

Ronald M. Fourney ◽

Rémy Aubin ◽

Kevin D. Dietrich ◽

Malcolm C. Paterson

Keyword(s):

Cell Lines ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Foreign Gene

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Determination of Copy Number of Target Carbohydrase Genes in the Penicillium verruculosum fungus Recombinant Strains

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-5-51-57 ◽

2019 ◽

Vol 35 (5) ◽

pp. 51-57 ◽

Cited By ~ 2

Author(s):

V.Yu. Kislitsin ◽

I.N. Zorov ◽

A.P. Sinitsyn ◽

A.M. Rozhkova

Keyword(s):

Copy Number ◽

Target Gene ◽

Gene Copy Number ◽

Gene Promoter ◽

Gene Copy ◽

Penicillium Verruculosum ◽

Penicillium Canescens ◽

Enzyme Preparations ◽

Recombinant Strains

The protein profiles of the culture liquids of recombinant Penicillium verruculosum strains that secreted an Aspergillus niger β-glucosidase (BGL series) or a Penicillium canescens xylanase A (XYLA series) have been previously analyzed. It was shown that the producers were divided into two groups according to the target protein content: 75-85% (1) and no more than 15% (2). It was established in this work that the copy number of the target gene bgll in the recombinant strains correlated with the β-glucosidase content in the BGL-F10 and BGL-F12 enzyme preparations. However, this proportion was violated in the direction of decreasing the xylanase A relative amount: the target enzyme content of 50% and 17% in the XylA3 and XylA4 preparations corresponded to 20-22 and 6 copies, respectively, of the xylA gene. We believe that this effect is a result of titration of positive transcription factors in the P. verruculosum XylA3 strain containing 20 to 22 copies of the cbhl gene promoter. Penicillium verruculosum, real-time PCR, gene copy number This work was partially supported by the Russian Foundation for Bsic Research (Project number: 18-29-07070).

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