Optimization of the conditions of the allele-specific primer extension reaction. (A) Comparison of four RT enzymes for their efficiency in incorporating labeled dNTPs and for their genotyping specificity in the array-based primer extension reaction. The MMLV RT is not as processibe as AMV and SuperScript because it yields lower signal intensities, but the MMLV RT showed the highest discrimination against misprimed extension. (MMLV = Moloney murine leukemia virus reverse transcriptase; AMV RT = avian myeloblastosis virus reverse transcriptase.) (B) Enhancement of the efficiency of nucleotide incorporation and genotyping specificity of the MMLV enzyme by increased reaction temperature and the presence of the disaccharide trehalose. Normalized signal intensities and signal intensity ratios are given on the y-axis.
