Agarose gels were incubated under soft agitation with depurination solution (0.25 M HCl, 15 min), denaturation solution (1.5 M NaCl and 0.5 M NaOH, 45 min), and neutralization solution (0.5 M tris and 1.5 M NaCl, 30 min). After overnight transfer, DNA was cross-linked (254 nm, 0.12 J) to a nylon membrane (0.45 mm; Pall Corporation). The membrane was prehybridized with Church buffer for 3 hours at 65°C, hybridized with 32P-labeled probes for 12 hours, rinsed with washing buffer (standard saline citrate, 0.1% SDS), and exposed to a phosphorimager screen.

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