The two forward AS primers, HFEW2 and HFEM, were 61 and 28 by long, respectively, and the complementary primer, HFECOM, was 26 by in length.
The first nucleotide difference (C or T) between AS primers HFEW2 and HFEM is located at the 3' terminal base.
The PCR reaction mixture contained the following in a final volume of 25 [micro]L: 50 ng of genomic DNA, PCR reaction buffer (10 mmol/L Tris-HCl, pH 8.8, 1.5 mmol/L Mg[Cl.sub.2], 50 mmol/L KCl, and 1 mL/L Triton X-100; Finnzymes), 5 mmol/L dNTP,1 U of DyNAzyme II DNA Polymerase (Finnzymes), 5 pmol of HFECOM and HFEW2, 20 pmol of HFEM, and 2.5 [micro]L of SYBR Green I 1:10 000 (Molecular Probes).
The results for the HFEW2, HFEM, and HFECOM primers with the GeneAmp 5700 Sequence Detection system are shown in Fig.
(23), it was necessary to use the longer AS primer (HFEW2) at a lower concentration than the shorter primer (HFEM).