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 transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (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 primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to 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 analyzed by computing densitometry. The ratio of competitor/target (Y-specific) 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 the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem 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 calculated to be around 50%.


CONCLUSIONS
This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.
Zhao
Y
Y
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
Lan
F
F
Gan
J
J
Yao
X
X
Reisner
Y
Y
D000818:Animals; D016026:Bone Marrow Transplantation / Q000276:immunology*; D017931:DNA Primers; D005260:Female; D008252:Macaca fascicularis; D008297:Male; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014019:Tissue Donors; D018183:Transplantation Chimera; D014184:Transplantation, Homologous; D014998:Y Chromosome
D017931:DNA Primers