Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.
BACKGROUND
Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens.


METHODS
We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction.


RESULTS
The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%.


CONCLUSIONS
OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.
Amicarelli
Giulia
G
Department of Biotechnology and Biosciences, University of Milano Bicocca, Milano, Italy.
Adlerstein
Daniel
D
Shehi
Erlet
E
Wang
Fengfei
F
Makrigiorgos
G Mike
GM
D000230:Adenocarcinoma / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D003062:Codon; D004247:DNA / Q000032:analysis*; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D017422:Sequence Analysis, DNA / Q000379:methods; D018631:ras Proteins
D003062:Codon; C117307:KRAS protein, human; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins