Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation

Background. Evaluation of the outcome of successful bone marrow transplanta tion (BMT) and in-depth studies of transplantation biology rely increasingl y on accurate detection of donor origin cells in the transplanted recipient s. This study describes a quantitative competitive polymerase chain reactio n (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. Methods. A competitor standard was generated via PCR using a mutagenic prim er that makes the competitor DNA 22 bp less than the wild type monkey Y-spe cific DNA. The mutated form can still be amplified by the primer pair for t he detection of monkey Y-specific DNA. A fixed amount of sample subjected t o chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were an alyzed by computing densitometry. The ratio of competitor/target (Y-specifi c) DNA for each sample pair was calculated. Results. Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of th e established quantitative PCR was found to be 25 pg of male DNA, which cor responds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34(+) stent cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calcula ted to be around 50%. Conclusions. This quantitative PCR assay offers both a high degree of speci ficity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.