Related Experiment Video
Updated: May 31, 2026
Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
Published on: August 23, 2024
Sensitive detection and quantification of the JAK2V617F allele by real-time PCR blocking wild-type amplification by
Cornelis J J Huijsmans1, Jeroen Poodt, Paul H M Savelkoul
1Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands. r.huysmans@jbz.nl
Abstract:
A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.
Insights
A new real-time PCR assay accurately quantifies the JAK2V617F mutation in myeloproliferative neoplasms. This method, blocking wild-type JAK2 amplification, offers sensitive detection for improved diagnosis and treatment monitoring.
Area of Science:
- Molecular Biology
- Hematology
- Oncology
Background:
- The JAK2 V617F mutation is a key driver in myeloproliferative neoplasms (MPNs).
- Accurate quantification of the JAK2 V617F allele burden is crucial for MPN diagnosis and treatment monitoring.
- Existing methods may lack sensitivity or linear range for precise quantification across diverse allele frequencies.
Purpose of the Study:
- To develop a sensitive and linear real-time PCR assay for detecting and quantifying the JAK2 V617F allele.
- To enable reliable quantification of JAK2 V617F across a broad range, from low to high allele burdens.
- To aid in the clinical and therapeutic management of MPNs associated with the JAK2 V617F mutation.
Main Methods:
- Development of a real-time PCR assay specifically targeting the JAK2 V617F mutation.
- Inhibition of wild-type JAK2 amplification using a peptide nucleic acid (PNA) oligonucleotide.
- Validation of the assay's linearity, reproducibility, and sensitivity using patient DNA diluted in wild-type DNA.
Main Results:
- The developed assay demonstrated linear amplification of JAK2 V617F from 0.05% to 100% allele burden.
- The assay exhibited acceptable reproducibility for quantification across the tested range.
- Sensitivity was determined to be 0.05%, with a low-level background signal observed in some healthy donors.
Conclusions:
- A sensitive and linear real-time PCR assay was successfully developed to detect and quantify the JAK2 V617F allele.
- The method's ability to quantify low-to-high allele burdens, by inhibiting wild-type amplification, is clinically valuable.
- This assay provides a reliable tool for predicting and diagnosing myeloproliferative neoplasms harboring the JAK2 V617F mutation.
