title: Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.
abstract: Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.
authors.affiliation: 1 Neurobiology of Disease Graduate Program, Mayo Graduate School , Rochester, Minnesota.
authors.forename: Han B
authors.initials: HB
authors.lastname: Lee
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Schwab
authors.affiliation: 3 Clinical and Translational Science Graduate Program, Mayo Graduate School , Rochester, Minnesota.
authors.forename: Alaa
authors.initials: A
authors.lastname: Koleilat
authors.affiliation: 3 Clinical and Translational Science Graduate Program, Mayo Graduate School , Rochester, Minnesota.|4 Medical Scientist Training Program, Mayo Graduate School , Rochester, Minnesota.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Ata
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors.forename: Camden L
authors.initials: CL
authors.lastname: Daby
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors.forename: Roberto Lopez
authors.initials: RL
authors.lastname: Cervera
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: McNulty
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors.forename: Hannah S
authors.initials: HS
authors.lastname: Bostwick
authors.affiliation: 1 Neurobiology of Disease Graduate Program, Mayo Graduate School , Rochester, Minnesota.|2 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.|3 Clinical and Translational Science Graduate Program, Mayo Graduate School , Rochester, Minnesota.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Clark
mesh_terms: D000483:Alleles; D000818:Animals; D003362:Cost-Benefit Analysis; D017931:DNA Primers; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D017422:Sequence Analysis, DNA; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; D029961:Zebrafish Proteins