Supplementary Information

Methods

Profiling of the length heteroplasmies

The polymorphisms and profiling of the length heteroplasmies (mtMS) in mtDNA from blood cells in the Korean population were investigated by performing sequence analysis and size-based PCR product separation by capillary electrophoresis of the mtDNA control region in 70 maternally unrelated donors. Fourteen samples came from each of the following age groups: cord blood; 1 to 20; 21 to 40; 41 to 60; over 61. The Institutional Review Board of the Chonnam National University Hwasun Hospital (Hwasun, Korea) approved the study protocols.

Cell preparation and total DNA extraction

The total blood leukocytes collected from the donors, pre–SCT recipients and post-SCT recipients were separated via density gradient centrifugation and washed twice in phosphate-buffered saline (PBS). The number of cells suspended in PBS was adjusted to 1 x 107 cells/mL. The total DNA was extracted with an AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejon, Korea). The extracted DNA was resuspended in a TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and was photometrically quantified.

Direct sequencing of the mtDNA control region

The control region (nucleotides 16024 to 16569 and 1 to 576) of mtDNA was amplified and sequenced using a set of designated primer pairs (Supplementary Table 2) and PCR conditions based on a published protocols.1, 2 Each amplified mtDNA product was purified using an AccuPrep PCR Purification Kit (Bioneer) and sequenced with a BigDye Terminator v3.1 Ready Reaction Kit (Applied Biosystems, Foster City, CA) and a ABI Prism 3100 Genetic Analyzer (Applied Biosystems).In order to determine the polymorphisms and mutations differing from the RCRS, the experimentally obtained mtDNA sequences were compared with the Revised Cambridge Reference Sequence (RCRS) (http://www.mitomap.org/mitomap/mitoseq.html) using the Blast2 program (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) and the database search tool, MitoAnalyzer (http://www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html, 2001). Potential artifacts were excluded by repeating the PCR amplifications from the original DNA samples once or twice, followed by sequencing as before.

Capillary electrophoresis (gene scan)

For the mtMS markers, five fluorescence-labeled primer pairs consisting of three control regions( 303 poly C , 16184 C, 514 (CA) repeats) and 2 published primer pairs in the coding regions (3566 poly C, 12385 poly C and 12418 poly A)3 shown in Supplementary Table 2 were used as candidate markers to monitor the extent of donor cell engraftment in the stem cell transplantation recipients. Each forward primer was labeled with a Hex (Bioneeer) fluorescent dye. The mtDNA products encompassing the poly-C tracts were amplified in a 50 μL reaction mixture containing 1 ng of the total DNA, 0.8 μM primer pairs, 400 μM of each dNTP, 1 U Taq DNA polymerase (TaKaRa LA Taq, Shiga, Japan), and 5 μL of a 10 x buffer and distilled water. The following PCR were used: initial denaturation at 96℃ for 1 min, followed by 32 cycles at 94 ℃ for 30 seconds, 58 ℃ for 30 sec, and 72 ℃ for 30 sec. After completing PCR, 1 μL of each PCR product and 0.5 μL of the size standard labeled with the fluorescent dye (Applied Biosystems) were added to 20 μL of deionized formamide. Denaturation was then carried out at 96 oC for 10 min, followed by a cooling step at -20 oC for 2 minutes. The denatured PCR products were separated via capillary electrophoresis using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) and Gene Scan Analysis Software (version 3.7, Applied Biosystems). Using this software, the fragment length of the specific PCR product as well as quantify its amount can be measured automatically by calculating the area under the fragment peak. Each sample was analyzed using at least two different PCR reactions with subsequent quantification in order to ensure accurate and reproducible results.

Six nuclear DNA STR markers as well as Amelogenin were used to evaluate the informativeness of the selected candidate mtMS markers (Supplementary Table 2). The method was carried out as described previously.4-6

TA cloning

To confirm heteroplasmy and the mixed nucleotide signals in the mtDNA control region, the PCR products were inserted directly into the pCR®2.1-TOPO® vector (TOPO TA cloning kit; Invitrogen) according to the manufacturer’s instruction. The recombinant plasmids isolated from 8 to 12 white colonies were then sequenced.7

In vitro mixing test for the determination of sensitivity

Blood cells from the donor and pre-transplant recipient were mixed at different dilutions in donor cell proportions of 1, 5, 10, 20, 30, 40 and 50% (Table 2). Samples of donor and recipient DNA underwent fluorescent-PCR using each minisatellite primer for mtMS and 303 poly C, and the nuclear DNA STR for D18S51 (Supplementary Fig. 2).

Statistical analysis

Wilcoxon rank sum tests was utilized for the determination of statistical differences in the donor cell percentage between the mixing test and the quantified donor chimerism (%) using 303 poly C mtDNA and nuclear D18S51 STR marker.

References

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3. Habano W, Nakamura S, Sugai T. Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome. Oncogene 1998;17:1931-1937.

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