IVS2nt-124 FasL polymorphism typing

used for the PCR were 5140Dir (5’-GCAGTTCAGACCTACATGAT- TAGGAT-3’) and 5378Rev (5’-CCAATTCTCACCTGTACCTTC-3’). The direct primer contains a mismatch (underlined and bold) that creates an artificial new restriction site in the PCR product; as a consequence the fragment can be cleaved by FokI in the presence of the allele G. For each PCR reaction in 25 ml, 100 ng of DNA template were added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl , 50 mM 2 KCl , 10 mM Tris Cl (pH 8.5) and 1 U of Taq polymerase (Promega). 2 The reaction was carried out in a PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 948C, 30 s at 628C, and 1 min at 728C. The first cycle was preceded by a step of denaturation at 948C for 6 min, and the last one was followed by a step of extension at 728C for 10 min. Ten ml of PCR product was digested with 1 U of FokI under recommended conditions and loaded on a 3% agarose gel. If allele G was present, the digestion generates a 210 bp fragment; conversely if A allele was present there was not any digestion and the 239 bp fragment remained intact.

Fas polymorphism typing

The 71377 polymorphism was analyzed in part by direct sequencing, and in part by allele specific amplification (ASA). Direct sequencing were carried out by amplification of the region spanning from base 71620 to 71276 respect to the ATG start codon of Fas, using primers Poly2Dir (5’-CCCCTTTTTTTCTCTCTTCCC-3’) and Poly2Rev (5’-

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IVS3nt169FasL polymorphism typing

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