APPLICATION OF A NEW PROTOCOL FOR NESTED PCR TO THE DETECTION OF MINIMAL RESIDUAL BCR ABL TRANSCRIPTS/

Nested PCR (NPCR), a two-step procedure in which the products of a fir st PCR using 'outer' primers are reamplified using 'inner primers', ha s been successfully used to test for the chronic myeloid leukemia (CML )-specific bcr-abl transcripts. A major drawback of the conventional n esting strategy is linked to the opening of the reaction tube between the two successive PCR reactions, giving a risk of contaminating the s econd mix with amplicons. In this paper, the application of a new prot ocol for NPCR without reopening the reaction tube between the two step s of the procedure is described for the research of residual leukemic cells in the peripheral blood of 14 CML patients treated by bone marro w transplantation (BMT) or interferon (IFN). This assay which is both highly specific and sensitive, offers several advantages over the use of conventional NPCR: it is more sensitive, faster and decreases the r isk of false-positive results. In addition, chemiluminescent detection of amplified DNA after transfer onto a nylon membrane, although compa rable with radioactive hybridization in terms of sensitivity and speed , is more advantageous in safety and convenience. In conclusion, this assay could be adapted to a number of clinical diagnostic uses.