Leader: Dr Helen
With the development
of a new family of saturating DNA dyes, high resolution melt curve
analysis (HRM) has been identified as a potentially
useful method of high throughput mutation scanning. Recent publications
suggest that HRM has a mutation detection sensitivity which is comparable
or superior to currently available pre-screening techniques. In 2006,
NGRL (Wessex) evaluated 3 machines capable of HRM analysis: RotorGene™ 6000
(Corbett Research), HR-1™ and 384 well LightScanner™ (both
from Idaho Technology). Eleven different amplicons were analysed:
seven amplicons were generated from the NGRL (Wessex) panel of generic
mutation detection control plasmids and 4 were generated from genomic
DNA: hMLH1 Exons 1, 7 & 13 and hMSH2 Exon 10. The amplicons varied
in size from 139 to 449bp and had GC contents ranging from 22 – 79%.
The types of mutations analysed included all possible point mutation
base substitutions and 1 and 2bp insertions and deletions.
Using the RotorGene™ 6000 (Corbett Research) PCR products were
amplified in the presence of the saturating dsDNA binding dye: LC
Green® Plus (Idaho Technology). 47 and 70 randomised samples
were amplified for each target plasmid and genomic DNA samples respectively
(624 samples in total, including controls) and amplification efficiency
was monitored in real-time. The same PCR products were analysed using
HRM on the RotorGene™ 6000, HR-1™ and 384 well LightScanner™ platforms.
Analysis of the RotorGene™ 6000 and HR-1™ melt curves
was undertaken manually by two operators and the LightScanner™ data
was analysed with the software supplied using high sensitivity settings.
Data were then unblinded and the sensitivity and specificity of mutation
detection were determined for each amplicon and platform.
A report of the evaluation and NGRL (Wessex) publications on
HRM can be found here.