Novel genetic and epigenetic alterations in ... - Ous-research.no

DiscussionDNA fragments to the column. In order to elute the fragments, increasing concentrations ofacetonitrile is flushed through the column. As the acetonitrile concentration increases, thebridging capabilities of the TEA ions decrease and the DNA fragments are released.Heteroduplexes, with mismatched base pairs, are eluted first followed by the homoduplexesdue to differences in melting temperature. Finally, the fragments pass through an UVdetector which detects the absorbance over time at 260nm[146] *** .The sensitivity of DHPLC is by large determined by temperature[147]. Included with theWAVE equipment is the Navigator TM software which based on fragment length and basecomposition predicts the optimal separation temperature, which is when 75% of thefragment is double stranded. If the temperature is too low, neither the homoduplex norheteroduplex fragments will be denatured, and will be eluted at the same time. Too hightemperature causes all fragments to be fully denatured before acetonitrile is added, againeluting the fragments at the same time. Only at optimal temperatures the heteroduplexes willdenature slightly before the homoduplexes, making mutation detection possible. Due tointra-fragment variations in base composition, PCR products longer than 200 bases usuallycontain two or more melting domains which mean that the product must be analyzed atseveral temperatures, reducing the throughput of the method. Other factors important forsensitivity are the resolution of the homoduplex and heteroduplex species as well as thepurity of the PCR product. With a good study design the sensitivity and specificity ofDHPLC is considered to be higher than 96%[146].In addition to DHPLC, a handful of sensitive mutation scanning methods exist, includingSSCP (Single-Strand Conformational Polymorphism)[148], TTGE (temporal temperaturegradient gel electrophoresis)[149], CSGE (Conformation Sensitive Gel Electrophoresis)[150]and DGGE (Denaturing Gradient Gel Electrophoresis)[151]. However, most, if not all ofthese, are clearly unsuitable when analyzing large samples series as these are gel basedmethods. In this setting a high throughput method would be more suitable, such asconformation-sensitive capillary electrophoresis (CSCE), MALDI-TOF based methods[152],and of course the new generation of ultra high-throughput sequencing systems. AlthoughDHLPC has an inferior throughput compared to these, with an estimated sensitivity higher*** Transgenomic web pages – http://www.transgenomic.com49