Τίτλος:
Bioluminometric assay for relative quantification of mutant allele burden: Application to the oncogenic somatic point mutation JAK2 V617F
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Unlike the inherited mutations, which are present in all cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair. To this end, we report the first bioluminometric assay for quantification of the allele burden and its application to JAK2 V617F somatic point mutation, which is a recently (2005) discovered molecular marker for myeloproliferative neoplasms. The method is performed in microtiter wells and involves a single PCR, for amplification of both alleles, followed by primer extension reactions with allele-specific primers. The products are captured in microtiter wells and detected by oligo(dT)-conjugated photoprotein aequorin. The photoprotein is measured within seconds by simply adding Ca2+. We have demonstrated that the percent (%) luminescence signal due to the mutant allele is linearly related to the allele burden. As low as 0.85% of mutant allele can be detected and the linearity extends to 100%. The assay is complete within 50 min after the amplification step. © 2009 American Chemical Society.
Συγγραφείς:
Tsiakalou, V.
Petropoulou, M.
Ioannou, P.C.
Christopoulos, T.K.
Kanavakis, E.
Anagnostopoulos, N.I.
Savvidou, I.
Traeger-Synodinos, J.
Περιοδικό:
Comprehensive Analytical Chemistry
Λέξεις-κλειδιά:
Luminescence signals; Molecular marker; Mutant alleles; Photoprotein aequorin; Photoproteins; Point mutations; Primer extension; Relative quantification; Residual disease; Specific primers, Calcium; Diagnosis; Wells, Amplification, aequorin; calcium ion; Janus kinase 2; molecular marker; photoprotein, allele; article; base pairing; bioluminescence; gene mutation; human; microtiter plate assay; minimal residual disease; myeloproliferative disorder; oncogene; point mutation; polymerase chain reaction; somatic mutation; statistical model, Aequorin; Alleles; Animals; Calcium; Cattle; DNA Primers; Humans; Janus Kinase 2; Linear Models; Luminescent Measurements; Magnesium; Point Mutation; Polymerase Chain Reaction
