Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction.
We fabricated and evaluated high-throughput kinetic thermal cyclers with 768-reaction capacity for kinetic polymerase chain reaction (kPCR)-based genotyping and kinetic reverse transcription (kRT)-PCR-based transcript quantitation. The system uses dye-based detection with ethidium bromide and a single DNA polymerase-based PCR or RT-PCR assay. Allele-specific detection of the two most common hereditary hemochromotosis mutant alleles, C282Y and H63D, was reliably measured by kPCR using human DNA templates as low as 10 genome equivalents per assay. Transcript profiling was performed for 16 yeast transcripts ranging in intracellular abundance over four orders of magnitude. Standard deviations of the PCR cycle threshold values determined from multiple kRT-PCR assays in three different instruments ranged from 0.11 to 0.97 PCR cycles and were reproducible, transcript specific, and instrument independent. The effects of the sin3, gal11, and snf2 knockout mutations on expression of 385 yeast genes were evaluated by kRT-PCR and compared to published values determined by high-density oligonucleotide array and/or microarray analysis for snf2 and sin3. The 768-reaction kinetic thermalcyclers, each with a capacity for more than a half million assays per year, are well suited to genomics applications such as single nucleotide polymorphism/disease association studies and genomewide transcription profiling where high sensitivity and accuracy are required.
Watson
Robert M
RM
Program in Core Research, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501, USA.
Griaznova
Olga I
OI
Long
Christopher M
CM
Holland
Michael J
MJ
D001483:Base Sequence; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D005800:Genes, Fungal / Q000235:genetics; D005838:Genotype; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018401:Sample Size
D012333:RNA, Messenger