DNA was extracted from the buccal cells using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Germany) according to the producer protocol. ACE genotyping PCR amplification of the polymorphic our site region of the ACE gene containing either the insertion (I) or dele-tion (D)

fragment was performed. Only one pair of primers (ACEfor: CTG GAG ACC ACT CCC ATC CTT TCT and ACErev: GAT GTG GCC ATC ACA TTC GTC AGA) was used to determine the ACE genotype, yielding amplification products of approximately 490 bp (for I allele) and 190 bp (for D allele). The 10 μl PCR consisted of: 1 μl DNA isolate; 0.5 U DNA recombinant Taq polymerase in buffer (pH = 8.0; Sigma, Germany); 1x PCR buffer (pH = 8.7; Sigma, Germany); 1.5 mM MgCl 2; 4 pM primer ACEfor and ACErev (Oligo, Poland) in TE buffer (pH = 8); 0.75 nM of each dNTP. The thermal-time PCR was as follows: initial denaturation at 94°C for 300 s, 30 cycles (denaturation

at 92°C for 60 s, primer annealing at 58°C for 60 s, chain extension at 72°C for 150 s) and final extension at 72°C for 360 s. The reaction was performed in two samples per isolate. Amplification products were visualized in UV light by using 1.5% agarose gels stained with ethidium bromide. ACTN3 genotyping The 290 bp fragment of exon 15 of the ACTN3 gene was amplified by PCR using the forward primer : 5′CTGTTGCCTGTGGTAAGTGGG-3′ and the reverse primer : 5′TGGTCACAGTATGCAGGAGGG-3′ as recommended by Mills et al. (2001). PCR reaction mix (total volume 10 μl) contained 1.5 mM MgCl2, 0.75 nM of each deoxynucleoside triphosphate (Novazym, Poland), 4 pM of each primer (Genomed, Poland), 0.5 U of Taq DNA polymerase (Sigma, Germany), and 1 μl (30–50 ng) of template DNA. After a first step

consisting of 95°C for 5 min, 35 cycles of amplification were performed by using denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s and a final cycle at 72°C for 10 min (Eynon et al., 2009). The amplified PCR fragments were subsequently digested with Dde I endonuclease (Fermentas, Lithuania) in a condition recommended by the supplier (Mills et al., 2001). The alleles 577R and 577X were distinguished by the presence Batimastat (577X) or absence (577R) of a Dde I restriction site. Digestion of PCR products of the 577X allele yields bands of 108, 97 and 86 bp, whereas digestion of PCR products of the 577R allele yields bands of 205 and 86 bp. The digested products were separated by 3% agarose gel electrophoresis, stained with ethidium bromide, and visualized in UV light. Statistical analysis The Hardy-Weinberg equilibrium (HWE) for ACE and ACTN3 genotypes was assessed separately in swimmers and control subjects with a χ2 test.