Analysis of β3 cDNA and theβ3 gene in patient Osaka-5.
(A) Nucleotide sequence analysis of β3 cDNA in patient Osaka-5. The β3 cDNAs from control or Osaka-5 platelets were amplified by RT-PCR. The amplified fragments were directly examined using Taq DyeDeoxy Terminator Cycle Sequencing kit. Samples were run and analyzed on an ABI 373A DNA sequencer. (B) Allele-specific restriction enzyme analysis. The region around exon 5 of the β3 gene was amplified by PCR using primers IIIaE5 and IIIaE6BspHI, followed by digestion with BspHI.BspHI digestion of the PCR products yields 205-bp and 24-bp fragments in normal allele. The A→C substitution abolished a restriction site for BspHI. The resulting fragments were electrophoresed in a 6% polyacrylamide gel; φX174 digested withHaeIII was used as a marker.
