Details:

Alternative Title:
BMC Med Genet
Personal Author:
Koontz, Deborah A ; Huckins, Jacqueline J ; Spencer, Antonina ; Gallagher, Margaret L
Koontz, Deborah A ; Huckins, Jacqueline J ; Spencer, Antonina ; Gallagher, Margaret L Less -
Description:
Background

Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1–2 are derived from CYP2A7, and exons 3–9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology.

Methods

A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow.

Results

Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics.

Conclusion

This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.


Subjects:
[+]
Alleles Aryl Hydrocarbon Hydroxylases Base Sequence Gene Frequency Genotype Humans Polymerase Chain Reaction Recombination, Genetic Sequence Alignment Sequence Analysis, DNA
Source:
BMC Med Genet. 2009; 10:80.
Document Type:
Journal Article
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:7422197d45052bb032cf8ebf874b008455a20165110e7e9446c0e2e79e981e43
Download URL:
https://stacks.cdc.gov/view/cdc/3411/cdc_3411_DS1.pdf
File Type:

Supporting Files

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  	1471-2350-10-80.nxml	txt
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