Materials. We have studied 343 DNA male DNA samples from the DNA bank at our institution, of which 211 were Macedonians, 111 Albanians and 21 Roma, Serbs or Turks. The study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia (R. Macedonia).

Methods. The set of 28 Y-SNP markers was grouped hierarchically into five multiplex polymerase chain reaction (mPCR)/primer extension reactions, so as to determine the most frequent haplogroups using one or two multiplexes (Figure 1). Most of the PCR and minisequencing primers have already been described [8], the remainder were designed by us.

Multiplex Polymerase Chain Reaction. The PCR primers were designed to give a variety of PCR fragment sizes, that allow their separation by polyacrylamide gel electrophoresis (Table 1 and Figure 2). The PCR multiplexes were performed in 25 L final volume, with 1X Reaction buffer, 300 M of dNTPs, 2 mM of MgCl₂, 2U of AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA, USA) and 10 ng of genomic DNA. Cycling conditions were 10 min. at 95C, then 35 cycles of 1 min. at 95C, 1 min. 15 s. at 58C, 3 min. at 72C and a final extension at 65°C for 15 min.

Multiplex Minisequencing. Before a single base extension (SBE), 1 L of PCR product was cleaned up with 0.5 L of ExoSAP-IT (USB Corporation, Clevelend, OH, USA) and incubated at 37C for 60 min. followed by 15 min. at 85C to inactivate the enzyme.

Multiplex single base extension reactions were performed in a 5 L final volume, combining 2 L of SNaPshot ready reaction mix (Applied Biosystems), 1.5 L of cleaned PCR product and 1.5 L extension primers. The minisequencing primers were 5' tailed with a poly-C sequence to produce extension products 21 to 51 nucleotides long to allow separation by capillary electrophoresis (Table 2). The cycling conditions were 10 s. at 96C, 10 s. at 50°C and 30 s. at 60C, for 25 cycles.

To remove unincorporated ddNTPs, the final products were incubated with 1U of shrimp alkaline phosphatase (USB Corporation) for 1 hour at 37C, and 15 min. at 85C to inactivate the enzyme.

Capillary Electrophoresis. The SNaPshot (Applied Biosystems) products were separated by capillary electrophoreis on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). Analysis of electropherograms was performed using the GeneScan 3.1 software (Applied Biosystems) and the size of fragments was determined on the basis of GeneScan-120 LIZ size standard (Applied Biosystems) (Figure 3).