Figure 4: Empirical evaluation of ThermoAlign using standard and long-range PCR to tile five genes.

The products from two additional genes amplified with standard PCR but not long-range PCR (as described in the text) are not shown. Labels indicate the chromosome number of the target locus, the forward primer start site and the expected size of the product. Details on each primer is available in Supplementary File S3. (a) Standard PCR products were quantified without post-PCR purification and approximately ≈7.5 ng were loaded into each well. For the two reactions that had no product, a volume equivalent to the average volume loaded was used. Multiplex reactions composed of primer pairs corresponding to each set for a given gene was loaded alongside the primers belonging to that same set. (b) Long-range PCR products from reactions without (−) and with (+) betaine. PCR products were quantified without post-PCR purification and ≈29 ng were loaded into each well. For the three reactions that had a no product, the same volume used for the corresponding betaine reaction was loaded into the well. For the negative control, the maximum volume used among all of the reactions was loaded into the well. The negative control was composed of master mix, primer pair TA_1_25390617_27_F and TA_1_25395472_24_R (Supplementary File S3) with no DNA template. Lanes with background smearing were associated with reactions that required a greater volume of the product be loaded to achieve a standardized amount of product across lanes.