Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis.
Allogeneic bone marrow transplant engraftment assays use polymorphisms in the human genome to determine the relative percentages of donor and recipient cells present in the recipient. We describe a novel posttransplant assay approach using single nucleotide polymorphisms (SNPs), the most common type of polymorphism in humans. Using samples of defined genotype, we used real-time polymerase chain reaction (PCR) and allele-specific fluorescent TaqMan probes to assay a SNP of the cytochrome P450 CYP2C9 gene. Standard curves of chimeric mixes showed a linear relationship between the ratio of two alleles and the ratio of their respective fluorophore emission, except for mixes with a low percentage (< 5%) of the less common allele. We validated the SNP real-time PCR assay by comparing it to Southern hybridization analysis, analyzing DNA mixes in a blinded fashion with both methods. The correlation between the two methods was high. We have produced a statistical model that varies allele frequency to predict how many SNPs would be required to produce a functional SNP panel. Additional development will be necessary to produce such a panel of highly informative SNPs for clinical use. A real-time PCR SNP assay may ultimately provide more accurate quantification and shortened turnaround time compared to current post-engraftment assays.
Department of Pathology, Johns Hopkins Hospital, Baltimore, USA.
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DH
Oliver
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Thompson
C A
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Griffin
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Eshleman
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases; D015139:Blotting, Southern; D016026:Bone Marrow Transplantation; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004247:DNA / Q000097:blood; D006085:Graft Survival; D006801:Humans; D015335:Molecular Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics
D015335:Molecular Probes; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase