Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis

Dwight H. Oliver, Richard E. Thompson, Constance A. Griffin, James R. Eshleman

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)202-208
Number of pages7
JournalJournal of Molecular Diagnostics
Volume2
Issue number4
DOIs
StatePublished - Nov 2000

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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