Primer design for PCR and sequencing in high-throughput analysis of SNPs.
To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.
Vieux
Ellen F
EF
Division of Dermatology, Washington University School of Medicine, St Louis, MO 63110, USA.
Kwok
Pui-Yan
PY
Miller
Raymond D
RD
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D030561:Databases, Nucleic Acid; D004867:Equipment Design; D005787:Gene Frequency; D015894:Genome, Human; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers