title: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection.
abstract: BACKGROUND
  The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity.
  
  
  METHODS
  We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2.
  
  
  RESULTS
  We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay.
  
  
  CONCLUSIONS
  QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.
authors.affiliation: Department of Pathology, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria 3002, Australia.
authors.forename: Giada V
authors.initials: GV
authors.lastname: Zapparoli
authors.forename: Robert N
authors.initials: RN
authors.lastname: Jorissen
authors.forename: Chelsee A
authors.initials: CA
authors.lastname: Hewitt
authors.forename: Michelle
authors.initials: M
authors.lastname: McBean
authors.forename: David A
authors.initials: DA
authors.lastname: Westerman
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005091:Exons; D005189:False Positive Reactions; D018922:HL-60 Cells; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics*; D018365:Neoplasm, Residual; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; C507924:JAK2 protein, human; D053614:Janus Kinase 2