Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping

Han B. Lee, Tanya L. Schwab, Alaa Koleilat, Hirotaka Ata, Camden L. Daby, Roberto Lopez Cervera, Melissa S. Mcnulty, Hannah S. Bostwick, Karl J Clark

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)425-435
Number of pages11
JournalHuman Gene Therapy
Volume27
Issue number6
DOIs
StatePublished - Jun 1 2016

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Alleles
Costs and Cost Analysis
Polymerase Chain Reaction
Fluorescent Dyes
Genotype
Restriction Fragment Length Polymorphisms
CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases
DNA-Directed DNA Polymerase
Reading
Molecular Biology
Animal Models
Hot Temperature
Fluorescence
Phenotype
Cell Line
Genes

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Lee, H. B., Schwab, T. L., Koleilat, A., Ata, H., Daby, C. L., Cervera, R. L., ... Clark, K. J. (2016). Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping. Human Gene Therapy, 27(6), 425-435. https://doi.org/10.1089/hum.2016.011

Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping. / Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; Mcnulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

In: Human Gene Therapy, Vol. 27, No. 6, 01.06.2016, p. 425-435.

Research output: Contribution to journalArticle

Lee, HB, Schwab, TL, Koleilat, A, Ata, H, Daby, CL, Cervera, RL, Mcnulty, MS, Bostwick, HS & Clark, KJ 2016, 'Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping', Human Gene Therapy, vol. 27, no. 6, pp. 425-435. https://doi.org/10.1089/hum.2016.011
Lee HB, Schwab TL, Koleilat A, Ata H, Daby CL, Cervera RL et al. Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping. Human Gene Therapy. 2016 Jun 1;27(6):425-435. https://doi.org/10.1089/hum.2016.011
Lee, Han B. ; Schwab, Tanya L. ; Koleilat, Alaa ; Ata, Hirotaka ; Daby, Camden L. ; Cervera, Roberto Lopez ; Mcnulty, Melissa S. ; Bostwick, Hannah S. ; Clark, Karl J. / Allele-specific quantitative PCR for accurate, rapid, and cost-effective genotyping. In: Human Gene Therapy. 2016 ; Vol. 27, No. 6. pp. 425-435.
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