A new allelic discrimination assay using locked nucleic acid-modified nucleotides (LNA) probes for detection of<i>JAK2</i>V617F mutation

Marková, Jana; Průková, Dana; Volková, Z; Schwarz, Jiřı́

doi:10.1080/10428190601137328

Cited by 8 publications

(9 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In 1114 patients, the JAK2 V617F mutation was screened by the real‐time RT‐PCR assay (wt and mutated alleles being discriminated by specific TaqMan probes positioned in between the common primers) as described by Marková et al . . In a minority of these cases, the commercial MutaScreen PCR kit from Ipsogen (Marseille, France), marketed currently by Qiagen (Düsseldorf, Germany), was used.…”

Section: Methodsmentioning

confidence: 99%

Thrombosis in thrombocythemic Ph‐ myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide

Schwarz

Ovesná

Cerná

et al. 2015

European J of Haematology

Self Cite

View full text Add to dashboard Cite

Controversies still exist regarding definition of the thrombotic risks in Ph-(BCR/ABL1-) myeloproliferative disorders with thrombocythemia (MPD-T). Platelet counts at diagnosis are currently not taken as a risk factor of thrombosis. In our cohort of 1179 patients with MPD-T, prospectively registered for anagrelide treatment, we found that the median platelet count prior to the thrombotic event was significantly higher than at time points without any ensuing thrombosis (453 vs. 400 9 10 9 /L, P < 0.001), albeit higher platelet counts at diagnosis tended to be connected with fewer thrombotic events (in contrast to WBC counts at diagnosis). The JAK2 V617F mutation predicted both arterial and venous events, while age >65 yr, hypertension, diabetes mellitus, smoking, elevated triglyceride and homocysteine levels predicted arterial events only. For venous events, the specific thrombophilic risk factors (factor V 'Leiden' and others), antiphospholipid antibodies, and elevated factor VIII levels played a major role. During anagrelide treatment (AE aspirin), we documented a decrease in both venous (6.7-fold) and arterial events (1.8-fold), while bleeding (mostly minor events) increased twofold compared to history. Our results suggest that keeping platelet counts at low levels may be a meaningful therapeutic measure to prevent thrombosis, although their counts at diagnosis lack any prognostic value.

show abstract

Section: Methodsmentioning

confidence: 99%

Thrombosis in thrombocythemic Ph‐ myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide

Schwarz

Ovesná

Cerná

et al. 2015

European J of Haematology

Self Cite

View full text Add to dashboard Cite

show abstract

“… 13 Our newly designed LNA probe, real-time PCR assay detects up to 0.1% of mutant DNA in a wild-type DNA background using gDNA in contrast to an earlier assay with an analytical sensitivity of 2% using complementary DNA. 19 This may be an improvement on previously published probes.…”

Section: Discussionmentioning

confidence: 84%

Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay

et al. 2018

View full text Add to dashboard Cite

The purpose of this study was to develop a real time polymerase chain reaction (PCR) assay for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Sanger sequencing and a newly developed locked nucleic-acid, real-time PCR assay were used to detect the JAK2 V617F mutation. There was 100% agreement between the sequencing and PCR analysis. Both assays were able to detect the mutation in all 24 of the 60 test specimens harbouring the mutation.

show abstract

“…Current strategies for realizing a suitable Δ T m,(MT–WT) therefore involve modifying oligonucleotides used as hydrolysis probes either by adding a 3′-terminal minor groove binder (MGB) ligand or by replacing nucleotides within the probe with their corresponding locked nucleic acid (LNA) analogue. , The latter approach can increase the difference in MT and WT duplex stabilities through the ability of LNA substitutions to decrease probe length while maintaining T m,MT , , and the generally greater energetic intolerance of LNAs to participate favorably in mismatched base pairs . LNA-bearing probes have therefore been shown to offer specific advantages when applied to the detection of either germline variants, including single-nucleotide polymorphisms (SNPs), ,, or acquired variants, including somatic point mutations (SPMs). − …”

mentioning

confidence: 99%

A New General Model for Predicting Melting Thermodynamics of Complementary and Mismatched B-Form Duplexes Containing Locked Nucleic Acids: Application to Probe Design for Digital PCR Detection of Somatic Mutations

et al. 2015

View full text Add to dashboard Cite

Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (BRAF V600E and KIT D816V) in a dPCR assay.

show abstract

A new allelic discrimination assay using locked nucleic acid-modified nucleotides (LNA) probes for detection ofJAK2V617F mutation

Cited by 8 publications

References 15 publications

Thrombosis in thrombocythemic Ph‐ myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide

Thrombosis in thrombocythemic Ph‐ myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide

Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay

A New General Model for Predicting Melting Thermodynamics of Complementary and Mismatched B-Form Duplexes Containing Locked Nucleic Acids: Application to Probe Design for Digital PCR Detection of Somatic Mutations

Contact Info

Product

Resources

About