A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity.
OBJECTIVE
The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity.


METHODS
We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis.


RESULTS
All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis.


CONCLUSIONS
To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.
Hiratsuka
Masahiro
M
Department of Pharmaceutical Sciences, Tohoku University Hospital, 980-8574, Sendai, Japan.
Narahara
Kaori
K
Kishikawa
Yukinaga
Y
Ismail Hamdy
Samar
S
Endo
Naomi
N
Agatsuma
Yasuyuki
Y
Matsuura
Masaki
M
Inoue
Tomoko
T
Tomioka
Yoshihisa
Y
Mizugaki
Michinao
M
D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D004351:Drug Resistance; D005838:Genotype; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D044402:Receptor, Serotonin, 5-HT2A; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*; D011985:Receptors, Serotonin / Q000235:genetics*
D017931:DNA Primers; D015342:DNA Probes; D004272:DNA, Mitochondrial; D044402:Receptor, Serotonin, 5-HT2A; D018343:Receptors, Adrenergic, beta-2; D011985:Receptors, Serotonin; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase