Detection of specific cancer gene mutations in liquid biopsies is the fastest and inexpensive way to reduce cancer deaths without imposing harm on the patients. Current procedures for detection of specific mutations persisting in very low numbers of copies, as compared to high level of the background wild type DNA, may not be sufficient for monitoring the target sequences. All existing methods rely on PCR amplification of the target DNAs in the reactions with known limitations due to non-specific annealing of primers which becomes critical at extremely low abundance of the targets.
Using standard primers for specific amplification of mutated DNA encounters the problem of non-specific amplification of WT templates. When copy number is very low, the standard number of 35-45 cycles in PCR may not be enough for amplification and detection of the target sequence. It is well known that increasing the number of cycles >45 in PCR may result in multiple non-specific artifacts.
FLAMPTM - Fimer-based extension method for enrichment of mutant templates by means of linear preamplification. Fimers are chemically modified primers. Specificity of Fimer annealing to the target template can be modulated by incorporation of several modified nucleotides within oligonucleotide sequence. Amplification takes place with long Fimers (e.g. 40-mers) only if all nucleotides are complementary to the template. Key point in thermocycling conditions is the very short duration of both annealing and extension steps. Linear amplification with Fimers can be carried out for 400 cycles or more and resulted in 2xn individual strands. After 400 cycles it would make up to 400 copies from each strand.