Abstract
Variable number tandem repeat (VNTR) and short tandem repeat (STR) markers of nuclear DNA are increasingly used for monitoring the engraftment of donor cells after stem cell transplantation (SCT). Mitochondria are the only organelles of animal cells other than the nucleus that contain DNA as well as their own machinery for RNA and protein synthesis. Mitochondrial DNA (mtDNA) is present in multiple copies (usually 103 to 104 copies per cell) (Blood2004;103:4466–77). We reported new markers using the analysis of mtDNA control region and microsatellites (mtMS) for monitoring donor cell engraftment in marrow transplant recipients. MtDNA control regions (nucleotide position (np) 16024 to 16569 and 1 to 576) and six mtMS (np 303–315 poly C, np 16184–16193 poly C, CA repeat starting at np 514, np 3566–3572 poly C, np 12385–12391 poly C and np 12418–12426 poly A) from total DNA samples from 25 sets of cases (donor, recipient and after SCT) were amplified using designated specific primers and PCR. We directly sequenced the control region which includes hypervariable regions, then carried out a qualitative and quantitative profiling mtDNA length heteroplasmies of six mtMS using size-based PCR product separation by capillary electrophoresis (ABI 3100 Genetic Analyzer and ABI Prism Genotyper version 3.1). The results were compared to those from six VNTR markers (D12S391, D1S80, F13A1, HUM RENA-4, HUM FABP2 and D18S51–5). MtDNA control region polymorphisms lead to the identification of an informative marker in 88% (22 cases) of all cases. Among six mtMS markers, the result of informativeness from np 303–315 and np 16184–16193 poly C mtMSs was 63% and 67% respectively. The combination of mtDNA control region direct sequencing, np 303–315 and np 16184–16193 poly C length heteroplasmies completely distinguished donor cells from recipients. In data from a typical mixing experiment for the determination of sensitivity, the detection limit (DL) of gene scan analysis in a mtDNA mixture was visible at 1% heteroplasmy in np 303–315 poly C mtMS marker; however DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. MtDNA control region sequencing and the length heteroplasmies of np 303–315 and np 16184–16193 poly C tracts can provide sensitive, accurate and quantitative determination of mixed chimerism after SCT.
Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA
