Determination of optimal conditions for detection of acute myeloid leukemia t(8;21) and t(15;17) translocations using RT-PCR.
Prognosis of patients with acute myeloid leukemia (AML) is not satisfactory. Long time remission can be achieved only in 25-40% children with AML. The aim of the study is to improve diagnosis of AML in cases characterized by specific chromosomal translocations t(8;21) and t(15;17). These translocations can be efficiently detected using reverse transcriptase-polymerase chain reaction (RT-PCR). To determine optimal conditions for detection of the t(8;21) and t(15;17) translocations in human cells, we performed a detail study of experimental conditions for RT-PCR on human cell lines NB-4 and KASUMI-1. For detection of t(8;21) using Pfx polymerase, the optimal PCR conditions included magnesium ion concentration in the range of 0.6 to 1 mM, 0.4 microM primer concentration, 5 degrees C annealing temperature and 1 microl of template cDNA. For detection of t(15;17) using the Pfx polymerase, the optimal conditions included 1 mM magnesium ion concentration, 0.6 microM primer concentration, 63 degrees C-66 degrees C annealing temperature and 2 microl of template cDNA. The results proved to be valid for clinical diagnostics of these chromosomal aberrations in blood and/or bone marrow samples of AML patients.
Fialová
M
M
Masaryk University, Faculty of Science, Brno, Czech Republic.
Sevcíková
S
S
Smarda
J
J
Michálek
J
J
D000328:Adult; D002412:Cations; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D006801:Humans; D007951:Leukemia, Myeloid / Q000175:diagnosis* / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry; D011237:Predictive Value of Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013696:Temperature; D014178:Translocation, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D002412:Cations; D017931:DNA Primers; D018076:DNA, Complementary; D008274:Magnesium