Formalin fixation and subsequent embedding in paraffin
tends to fragment and cause adducts in the DNA that can make analysis challenging . 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 successfully used to analyse and detect mutations in DNA derived LY2606368 order from formalin-fixed paraffin-embedded tumours (Niraparib cell line FF-PETs) for many years. It is a well-established method, widely available and relatively inexpensive to use [4, 5] and can detect any mutation in the sequence being analysed. DNA sequencing is often quoted
as the ‘gold standard’ for DNA sequence analysis . However, sequencing is not exquisitely sensitive. A mutation must be present in approximately 20% of the sample to be readily detected [7, 8]. Studies in colorectal cancer have found the percentage mutation in a tumour sample to be as low as 6%, significantly lower than sequencing is able to detect . Given the heterogeneity of tumours  the percentage is possibly even lower in some tumour biopsy specimens. We have extensive experience in the development and use of the allele specific polymerase chain reaction (PCR)-based method ARMS™ selleck products (Amplification Refractory Mutation System) . These assays are sensitive, routinely being able to detect at least 1% mutant in a normal DNA background, and are quick and easy to use. This PCR-based Reverse transcriptase method can be further enhanced by the ability to analyse the results in a real-time, closed-tube format by incorporating fluorescent probes such as TaqMan , Scorpions , Molecular Beacons  or intercalating fluorescent dyes such as Yo-Pro  or Sybr green , which eliminates PCR product contamination and reduces the time to generate
results. They perform well on FF-PET-derived DNA and their sensitivity makes them ideal for the analysis of heterogeneous tumour samples. Unlike sequencing, ARMS assays only detect the mutations they were designed to interrogate. However, this could be considered an advantage in a clinical setting so that decisions on treatment or patient-outcome results are based only on known, clinically validated mutations. We have evaluated three real-time ARMS assays in melanoma tumour samples: BRAF 1799T>A [this includes V600E and V600K], NRAS 182A>G [Q61R] and 181C>A [Q61K], and two real-time ARMS assays in non-small-cell lung cancer (NSCLC) samples: EGFR 2573T>G [L858R] and 2235-2249del15 [E746-A750del], for the analyses FF-PET DNA and compared the results to DNA sequencing of the exons containing mutation hot-spots for these genes (BRAF exon 15, NRAS exon 1, EGFR exons 18-21).