Dinucleotide repeat polymorphism at the D11S490 locus.

PCR Conditions: PCR reactions are carried out in a total volume of 25 fi\ containing: 25 ng genomic DNA, 5 pmole of each primer, 1.5 mM MgCl2, 200 jiM dNTPs, 50 mM tris-Cl, 20 mM (NH^SO,,, pH 9, 0.25 mM spermidine and 0.6 units of ReplinaseTM (NEN/Dupont). Amplification was performed in 96 well plates in a BiosyclerTM oven (Bios Corp., New Haven, CT) for 32 cycles. We used a 4 temperature cycle with denaturation at 93°C for 60 seconds, pre-annealing at 53°C for 10 sec, annealing at 48°C for 60 seconds and extension at 72°C for 15 seconds. A 3 minute ramp was programmed for the transition from 53°C to 48°C; all other ramps were programmed to be as brief as possible. PCR products were resolved on DNA sequencing gels, capillary-blotted onto Hybond N+TM nylon membranes (Amersham) and revealed by probing with 5'[P] labeled (CA)10 oligomer.