Fig. 2.
Identification of the genetic defect responsible for Osaka-12.

Identification of the genetic defect responsible for Osaka-12.

(A) Nucleotide sequence analysis of αIIb cDNA from Osaka-12. Platelet αIIb mRNA was amplified by RT-PCR. Nucleotide sequence of the amplified fragments was determined by using Taq DyeDeoxy Terminator Cycle Sequencing kit and an ABI 373A DNA sequencer. (B) Allele-specific restriction enzyme analysis of αIIb cDNA. The region around exon 4 of the αIIb mRNA was amplified by PCR, followed by digestion with ScaI. ScaI digestion of the PCR products yielded 517-bp, 264-bp, and 316-bp fragments in the healthy allele. The T>C substitution abolished one of the restriction sites forScaI. Resultant fragments were electrophoresed in a 1.5% agarose gel. Marker indicates 100-bp DNA ladder. (C) Allele-specific restriction enzyme analysis of αIIb genomic DNA. The region around exon 4 of the αIIb gene was amplified by PCR using primers IIb3945S and IIbE4, followed by digestion withScaI. ScaI digestion of the PCR products yielded 241-bp and 75-bp fragments in the healthy allele. The T>C substitution abolished the restriction site for ScaI. Resultant fragments were electrophoresed in a 6% polyacrylamide gel. Marker indicates φX174 digested with HaeIII.

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