Protocols for the analysis of HGO mutations and polymorphisms
Microsatellite analysis:
Analysis of polymorphisms at the HGO-1 (D3S4496), HGO-2 (D3S4497) and HGO-3 (D3S4556) was performed by PCR, using total human genomic DNA, as described elsewhere. In brief, amplification was performed in a total volume of 20 microliter, containing: 100 ng of genomic DNA, 5pmol of each primer (Click here to see primer pairs), 1 u. Taq polymerase (Perking-Elmer Cetus), 250 micromolar each dATP, dGTP, and dTTP, 10 micromolar dCTP, 1microCi a -[32P] dCTP at 300 Ci mmol-1, 1 mM MgCl2 (or 1.25 mM in the case of HGO-3), 50 mM KCl, and 10 mM Tris-HCl (pH 8.3). PCR conditions were one cycle at 94ēC for 5 min., followed by two cycles at 94ēC for 1 min., 60ēC for 1 min., and 72ēC for 1 min, and by 30 cycles (or 25, in the case of HGO-3) at 94ēC for 45 s. And 60ēC for 40 s., ending with one cycle at 72ēC for 3 min. Samples were resolved on 6% polyacrylamide sequencing geles and were exposed on Kodak XAR films, with intensifying screens at –70ēC, for 2-12 h.
SSCP Analysis:
SSCP analysis was performed by PCR, using total genomic DNA, as described elsewhere (Orita et al, 1989). In brief, amplification was performed in a total volume of 10 microliter, containing: 100 ng of genomic DNA, 12.5pmol of each primer (Click here to see primer pairs), 1 U. Taq polymerase (Perking-Elmer Cetus), 250 micromolar each dATP, dGTP, and dTTP, 10 micromolar dCTP, 1 microCi a -[32P] dCTP at 300 Ci mmol-1, 1.5 mM MgCl2 (or 1.25 mM in the case of HGO-3), 50 mM KCl, and 10 mM Tris-HCl (pH 8.3). PCR conditions were one cycle at 94ēC for 3 min., followed by 30 cycles at 94ēC for 1 min., 60ēC for 1 min., and 72ēC for 1 min., ending with one cycle at 72ēC for 3 min. Samples were resolved on 8% or 10% polyacrylamide non denaturing geles (39:1 acrylamide:bisacrylamide), and were exposed on Kodak XAR films, with intensifying screens at –70ēC, for 2-10 h.
PCR Amplification of HGO Exons and Sequencing:
Exons of the HGO gene were amplified from genomic DNA by use of the specific primers derived from the 5´ and 3´ intronic sequences (to see sequences of primer pairs click the corresponding HGO exon in the table of mutations and polymorphisms). The annealing temperature of all primer pairs was 60ēC. The corresponding PCR products were purified by agarose gel electrophoresis and extraction with Wizard PCR Preps DNA purification system (Promega). Direct sequencing of PCR products was performed with a dye-terminator cycle-sequencing kit (Perkin-Elmer) using Taq FS DNA polymerase. Sequences were resolved on an ABI PRISM 377 automatic sequencer.