TUF (C + G rich) sequences impair the analysis of neighbouring DNA regions. The lower image shows a restriction map surrounding the ‘test’ fragment of PRT assay A2n13, including a 1100 bp region with 73% C + G content (green box). The graph above shows average test:reference product ratios that were run in triplicate, with maximum and minimum plotted as error bars. ‘Control’ indicates the use of undigested DNA. Remaining columns show the ratios produced upon pre-digesting with the indicated restriction enzymes. The absolute degree of reference fragment amplification did not vary significantly across these treatments. Treatments that break the DNA to physically separate the test fragment from the C + G rich sequence clearly provide the best improvement in test fragment amplification efficiency. This reaches 1.43, which is the theoretical maximum assuming exactly equal molar amplification of test and reference amplicons (as indicated by the dotted line).
Veal et al. BMC Genomics 2012 13:455 doi:10.1186/1471-2164-13-455