RT-PCR plus restriction endonuclease analysis of peripheral blood reticulocyte RNA from a normal subject and from 2 β15 carriers.

(A) Schematic representation of the protocol. The reticulocyte mRNA was reverse transcribed, originating cDNA fragments of 580 nt. Boxed areas represent human β-globin gene exons, and AUG and UAA represent translation initiation and termination codons, respectively. β-cDNA was amplified, producing DNA fragments of 496 bp. The localization ofAspHI and ApaLI restriction sites is indicated. The AspHI/ApaLI polymorphic restriction enzyme site, located at codon 2, is indicated by an asterisk. In the β15 carriers, the mutated allele is linked to allele 1 at codon 2. (B) Representative ethidium bromide-stained agarose gel electrophorogram of normal undigested DNA (lane 1), AspHI-digested normal DNA (lane 2), ApaLI-digested normal DNA (lane 3), undigested DNA from 2 β15 carriers (lanes 4 and 7, respectively),AspHI-digested DNA from 2 β15 carriers (lanes 5 and 8, respectively), and ApaLI-digested DNA from 2 β15 carriers (lanes 6 and 9, respectively). Fragment length is indicated on the right.