title: Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms.
abstract: BACKGROUND
  Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR).
  
  
  METHODS
  Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing.
  
  
  RESULTS
  Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing.
  
  
  CONCLUSIONS
  Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations.
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Taira
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.forename: Akemi
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Akane
authors.initials: A
authors.lastname: Sueki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Koeda
authors.forename: Fumio
authors.initials: F
authors.lastname: Takagi
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Sugano
authors.forename: Takayuki
authors.initials: T
authors.lastname: Honda
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004847:Epithelial Cells / Q000737:chemistry; D056426:Genetic Loci; D005838:Genotype; D060005:Genotyping Techniques; D064368:Healthy Volunteers; D006801:Humans; D057230:Limit of Detection; D009061:Mouth Mucosa / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004247:DNA
keywords: AS-PCR; Allele specific-PCR; Droplet-PCR; HRM analysis; PB; SNP; Single nucleotide mutation; Single nucleotide polymorphism; allele specific-polymerase chain reaction; droplet-AS-PCR; droplet-allele specific-polymerase chain reaction; high resolution melting analysis; peripheral blood; single nucleotide polymorphism