A DNA Real-time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia

Publication date: Available online 29 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Paul A. Bartley , Susan Latham , Bradley Budgen , David M. Ross , Elizabeth Hughes , Susan Branford , Deborah White , Timothy P. Hughes , Alexander A. Morley The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. PCR primers were chosen from a library of presynthesized and pretested BCR (n = 19) and ABL1 (n = 568) primers. BCR-ABL1 sequences were quantified relative to BCR sequences in 521 assays on 266 samples from 92 patients. For minimal residual disease detectable by DNA qPCR and RT-qPCR, DNA qPCR gave similar minimal residual disease results as RT-qPCR but had better precision at low minimal residual disease levels. The limit of detection of DNA qPCR depended on the amount of DNA assayed, being 10−5.8 when 5 μg was assayed and 10−7.0 when 80 μg was assayed. DNA qPCR may be useful and practical for monitoring the increasing number of patients with minimal residual disease around or below the limi...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research