Optimization of a Low-Cost, Sensitive PNA Clamping PCR Method for JAK2 V617F Variant Detection
| Autor: | Oriana Catapano, Concetta Giancola, Luigi Petriccone, Ferdinando Frigeri, Raffaele Di Francia, Annunziata Cummarro, Stefania Crisci, Tommaso Muto, Antonio Pinto |
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| Přispěvatelé: | Di Francia, Raffaele, Crisci, Stefania, Muto, Tommaso, Giancola, Concetta, Petriccone, Luigi, Catapano, Oriana, Cummarro, Annunziata, Pinto, Antonio, Frigeri, Ferdinando |
| Rok vydání: | 2020 |
| Předmět: |
Male
Peptide Nucleic Acids 0301 basic medicine Serial dilution Cost-Benefit Analysis DNA Mutational Analysis PNA/DNA Computational biology Biology Polymerase Chain Reaction Sensitivity and Specificity DNA sequencing 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cell Line Tumor medicine CRITERIA Humans ASSAYS Genotyping Myeloproliferative neoplasm Myeloproliferative Disorders Peptide nucleic acid MOLECULAR DIAGNOSTICS DNA General Medicine Gold standard (test) Janus Kinase 2 medicine.disease Minor allele frequency 030104 developmental biology chemistry 030220 oncology & carcinogenesis Mutation Female NEOPLASMS |
| Zdroj: | The Journal of Applied Laboratory Medicine. 5:643-655 |
| ISSN: | 2475-7241 2576-9456 |
| DOI: | 10.1093/jalm/jfaa041 |
| Popis: | Background The JAK2 V617F variant is diagnostic for myeloproliferative neoplasms, a group of clonal disorders of hematopoietic stem and progenitor cells. Although several approaches have been developed to detect the variant, a gold standard diagnostic method has not yet been defined. We describe a simple, fast, and cost-effective PCR-based approach that enhances test specificity and sensitivity by blocking the amplification of the large excess of wild-type DNA. Methods The method involves using an oligo peptide nucleic acid (PNA) perfectly matching its corresponding DNA sequence. The PCR protocol was optimized by collecting a detailed thermodynamic data set on PNA-DNA wild-type duplexes by circular dichroism melting experiments. The specificity and sensitivity of PNA clamping PCR were assessed by genotyping 50 patients with myeloproliferative neoplasm who carried the JAK2 V617F variant and 50 healthy donors. Results The optimized protocol enabled selective amplification of the variant alleles, achieving maximum sensitivity (100%) and specificity (100%). Analytical sensitivity was 0.05% of variant alleles as assessed by serial dilutions of DNA from the HEL cell line (which carries the JAK2 V617F variant) mixed to wild-type DNA from healthy donors. The JAK2 V617F variant test performed according to this method has better diagnostic performance than its 2 main PCR-based competitors, at much lower cost. Conclusions High sensitivity and specificity and cost-effectiveness make PNA clamping PCR a useful testing platform for the detection of minor allele variants in small-scale diagnostic laboratories. It promises to improve patient care while enabling significant healthcare savings. |
| Databáze: | OpenAIRE |
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