Time-saving multiplex detection of single nucleotide polymorphisms by ultrasensitive DNA microarray.
Rapid and multiplex detection system using an ultrasensitive DNA microarray was developed and utilized for the analysis of six pharmacokinetically relevant single nucleotide polymorphisms (SNPs) (MDR1-C1236T, MDR1-G2677TA, MDR1-C3435T, CYP3A5-A6986G, CYP2C19-G681A, CYP2C19-G636A) from blood samples derived from liver transplant patients. The SNP detection system is comprised of three processes: multiplex PCR, single base extension with fluorescently labelled di-deoxy-nucleotides and detection by DNA microarray. The entire workflow of this system completes within 5 h. The final genotype call was obtained statistically by Mahalanobis distance which was calculated from the bi-coloured fluorescent signals detected by the microarray. In order to detect the six SNPs, this system required only 50 copies of genomic DNA, and the obtained detection calls completely matched with the results by the sequencing-based genotyping method. With the high sensitivity and rapid processing, our SNP detection system utilizing ultrasensitive microarray is a promising device applicable for diagnostic utility.
Ichikawa
Makiko
M
Toray Industries, Inc., New Frontiers Research Laboratories, 6-10-1, Tebiro, Kamakura, Kanagawa 248-0036, Japan. makiko_ichikawa@nts.toray.co.jp
Miwa
Keishi
K
Yamasaki
Tomo
T
Nakagawa
Izumi
I
Takizawa
Satoko
S
Masuda
Satohiro
S
Inui
Ken-ichi
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D005821:Genetic Techniques; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A