title: Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.
abstract: The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.
authors.affiliation: Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. nishida-75@umin.ac.jp
authors.forename: Nao
authors.initials: N
authors.lastname: Nishida
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Mawatari
authors.forename: Megumi
authors.initials: M
authors.lastname: Sageshima
authors.forename: Katsushi
authors.initials: K
authors.lastname: Tokunaga
mesh_terms: D001681:Biological Assay / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004586:Electrophoresis; D056426:Genetic Loci / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase