A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples

We have evaluated five real-time ARMS assays: BRAF 1799T>A, [this includes V600E and V600K] and NRAS 182A>G [Q61R] and 181C>A [Q61K] in melanoma, EGFR 2573T>G [L858R], 2235-2249del15 [E746-A750del] in non-small-cell lung cancer, and compared the results to DNA sequencing of the mutation 'hot-spots' in these genes in formalin-fixed paraffin-embedded tumour (FF-PET) DNA.The ARMS assays maximised the number of samples that could be analysed when both the quality and quantity of DNA was low, and improved both the sensitivity and speed of analysis compared with sequencing. ARMS was more robust with fewer reaction failures compared with sequencing and was more sensitive as it was able to detect functional mutations that were not detected by DNA sequencing. DNA sequencing was able to detect a small number of lower frequency recurrent mutations across the exons screened that were not interrogated using the specific ARMS assays in these studies.ARMS was more sensitive and robust at detecting defined somatic mutations than DNA sequencing on clinical samples where the predominant sample type was FF-PET.The molecular analysis of tumours has become increasingly important in recent years, particularly to aid the choice of drug therapy [1,2]. Assays to evaluate clinical samples, particularly if the results are used to determine treatment regimens, need to be rapid, precise and specific. However, the processes used to prepare tumours for standard pathology analysis for diagnosis, while preserving the tissue architecture, have a detrimental effect on DNA and RNA. Formalin fixation and subsequent embedding in paraffin tends to fragment and cause adducts in the DNA that can make analysis challenging [3]. In addition, tumour specimens are heterogeneous. They can contain surrounding and infiltrating normal cells, and not all tumour cells are identical. Analysis methods must therefore also be sensitive.DNA sequencing is one of the most widely used methods for analysing DNA and has been s