scholarly journals Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry

2002 ◽  
Vol 30 (5) ◽  
pp. 22e-22 ◽  
Author(s):  
S. Sauer
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

scholarly journals MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms

2007 ◽  
Vol 53 (1) ◽  
pp. 24-33 ◽  
Author(s):  
Julia K Blievernicht ◽  
Elke Schaeffeler ◽  
Kathrin Klein ◽  
Michel Eichelbaum ◽  
Matthias Schwab ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphisms ◽  
Direct Sequencing ◽  
Plasma Concentrations ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Ms ◽  
Hepatic Expression ◽  
Maldi Tof ◽  
Tof Ms

Abstract Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. Results: Five multiplex assays were developed, comprising the following 15 SNPs: −82T→C (*22); 86G→C (R29T, *17); 136A→G (M46V, *11); 296G→A (G99E, *12); 415A→G (K139E, *8, *13); 419G→A (R140Q, *14); 516G→T (Q172H, *6, *7, *9, *13, *19, *20), 547G→A (V183I); 769G→A (D257N); 785A→G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T→C (I328T, *16, *18); 1006C→T (R336C, *19); 1172T→A (I391N, *15); 1282C→A (P428T, *21); 1459C→T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. Conclusions: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.

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

2007 ◽  
Vol 53 (5) ◽  
pp. 933-939 ◽  
Author(s):  
Ashish Misra ◽  
Jun-Yan Hong ◽  
Sobin Kim
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Affinity Purification ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Base Extension

Abstract 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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