Optimization of a Low-Cost, Sensitive PNA Clamping PCR Method for JAK2 V617F Variant Detection
© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:5 |
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| Enthalten in: |
The journal of applied laboratory medicine - 5(2020), 4 vom: 01. Juli, Seite 643-655 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Di Francia, Raffaele [VerfasserIn] |
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| Links: |
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| Themen: |
EC 2.7.10.2 |
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| Anmerkungen: |
Date Completed 19.07.2021 Date Revised 19.07.2021 published: Print Citation Status MEDLINE |
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| doi: |
10.1093/jalm/jfaa041 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a © American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com. | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a PNA clamping PCR | |
| 650 | 4 | |a thermodynamic PNA-DNA duplex | |
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| 700 | 1 | |a Muto, Tommaso |e verfasserin |4 aut | |
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| 700 | 1 | |a Cummarro, Annunziata |e verfasserin |4 aut | |
| 700 | 1 | |a Pinto, Antonio |e verfasserin |4 aut | |
| 700 | 1 | |a Frigeri, Ferdinando |e verfasserin |4 aut | |
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