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Among the most important genes up-regulated in ECs in this condition (36) are those that denote EC activation, (45) namely, IL-8, MCP1, VCAM1, PAI1, Gro-[alpha], and E-selectin.
We present here a single-step method for genotyping of the 4G/5G polymorphism of the PAI1 gene that uses rapid-cycle PCR and subsequent analysis with resonance energy transfer probes on a thermal cycler with real-time fluorescence monitoring (15).
The primer 5'-AGCCAGACAAGGTTGTTGACAC-3' (nucleotides -741 to -720, relative to the transcription start site) and the primer 5'-CAGAGGACTCTTGGTTTTCCC-3' (nucleotides -628 to -607, relative to the transcription start site) were used to amplify, respectively, a 134- or a 135-bp fragment of the human PAI1 gene that harbors the polymorphic site 675 nucleotides upstream from the start of transcription (GenBank locus HSPAI11) (17).
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