Why does the DNA MLPA protocol start with 5 min. incubation at 98 °C?
The DNA detection/quantification protocol starts with 5 minute incubation at 98 °C both to denature the DNA and to generate fragments with an average size of 10 Kb. This fragmentation will also reduce the viscosity of chromosomal DNA solutions. This heat treatment is not included in the RT-MLPA protocol for mRNA analysis as the cDNA fragments to which the RT-probes hybridise are already very small. Following addition of MLPA buffer and probes, samples are again heated for 1 minute at 95°C in order to reduce secondary structure of probes and to dissolve probe aggregates.
