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Clinical Chemistry 53: 933-939, 2007. First published March 23, 2007; 10.1373/clinchem.2006.080739
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(Clinical Chemistry. 2007;53:933-939.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Multiplex Genotyping of Cytochrome P450 Single-Nucleotide Polymorphisms by Use of MALDI-TOF Mass Spectrometry

Ashish Misra1, Jun-Yan Hong3 and Sobin Kim2,a

Departments of1 Chemical and Biochemical Engineering and 2 Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ.
3 School of Public Health/Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey, Piscataway, NJ.

aAddress correspondence to this author at: Biomedical Engineering, Rutgers University, 599 Taylor Rd., Rm. 207, Piscataway, NJ 08854. Fax 732-445-3753; e-mail sobinkim{at}rci.rutgers.edu.

Background: Polymorphisms in cytochrome P450 (CYP450) genes contribute to interindividual differences in the metabolism of xenobiotic chemicals, including the vast majority of drugs, and may lead to toxicity and adverse drug reactions. Studies on these polymorphisms in research and diagnostic settings typically involve large-scale genotyping and hence require high-throughput assays.

Methods: We used the previously developed solid-phase capture–single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes.

Results: We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13.

Conclusions: The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.




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