Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.
Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.
Labuda
D
D
Centre de Cancérologie Charles-Bruneau, Centre de recherche de l'Hôpital Sainte-Justine, Montréal, Quebec, Canada. Damian.Labuda@Umontreal.ca
Krajinovic
M
M
Richer
C
C
Skoll
A
A
Sinnett
H
H
Yotova
V
V
Sinnett
D
D
D001191:Arylamine N-Acetyltransferase / Q000032:analysis* / Q000235:genetics; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis* / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D007527:Isoenzymes; D009841:Oligonucleotides; D019363:Cytochrome P-450 CYP1A1; D019389:Cytochrome P-450 CYP2D6; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1