Practice question

RFLP genotyping requires a probe that actually spans the variable restriction site to differentiate alleles reliably on Southern blot. Coding probes hybridizing far away may detect fragments independent of polymorphism, missing variation. Intron or flanking probes outside restriction region might not cover site-containing junction. A polymorphic probe designed across the restriction site junction recognizes sequences that are cleaved in one allele but remain intact in another, yielding distinct band sizes. Such allele-specific hybridization enables discrimination of homozygous normal, homozygous mutant, and heterozygous individuals, essential for linkage analysis and disease diagnosis applications.