Bioluminometric assay for relative quantification of mutant allele burden: application to the oncogenic somatic point mutation JAK2 V617F.
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 Ca(2+). 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.
Tsiakalou
Vaya
V
Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
Petropoulou
Margarita
M
Ioannou
Penelope C
PC
Christopoulos
Theodore K
TK
Kanavakis
Emmanuel
E
Anagnostopoulos
Nikolaos I
NI
Savvidou
Ioanna
I
Traeger-Synodinos
Jan
J
D000331:Aequorin / Q000378:metabolism; D000483:Alleles; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics* / Q000378:metabolism; D016014:Linear Models; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D000331:Aequorin; D053614:Janus Kinase 2; D008274:Magnesium; D002118:Calcium