Prevention of product carry-over by single tube two-round (ST-2R) PCR: application to BCR-ABL analysis in chronic myelogenous leukemia

摘要

False positive results in two-round nested PCR reactions most frequently originate from carry-over of amplified products. It is desirable, therefore, to avoid handling of the first round PCR product prior to amplification with internal primers. Ideally, both amplifications steps should be performed in a single tube without having to open it between first and second PCR round. Earlier approaches to this task have not entirely eliminated problems such as interference of external primers during the second round of amplification. This may result in the formation of spurious products requiring subsequent hybridization to ensure specific detection of target sequences. Here we present a simple two-round PCR protocol which permits highly sensitive and specific amplification in a single tube, and demonstrate its application to the detection of leukemic cells carrying a BCR-ABL rearrangement in patients with chronic myelogenous -leukemia (CML).