(327d) Microchip-Based Electrophoretic Analysis of Low Abundant Unknown Mutations Using Site-Specific Nicking and Ligation Enzymes

The onset of colorectal cancer has been linked to a host of well characterized point mutations that occur primarily within codons 12 and 13 of the K-ras oncogene as well as unknown sporadic p53 tumor suppressor gene mutations. The presence of these low abundance mutations, which are masked in normal genomic DNA sequences, can be used as biomarkers for early detection or monitoring disease progression. Two strategies conducive to microchip electrophoretic separations have been implemented, which make use of enzymes that recognize PCR amplified mutational sites for the detection of both known and unknown mutations; the ligase detection reaction (LDR) and the endonuclease V (EndoV) nicking/resealing assays. The former, LDR, is based on an allele-specific thermostable ligation of two complementary primers, which flank a point mutation and hybridize to target DNA.[1] The latter, EndoV, effectively nicks DNA at mutation sites within generated heteroduplexes of two fluorescently labeled sequences in addition to some miscleavages, which can be subsequently resealed using ligation in real-time to reduce non-target signals.[2] High aspect ratio microstructures (HARM) fabricated using mold inserts generated via LiGA were hot embossed into poly(methyl methacrylate), PMMA, substrates. Separations of multiple dye-labeled LDR products and discriminating primers (~20-65 bp) with normal-to-mutant DNA ratios of 1:100 were achieved using assembled microchips filled with replaceable LPA matrices and detected with an in-house built laser-induced fluorescence (LIF) system. Using two color excitation (532, 780 nm), dye-labeled EndoV cleaved top and bottom strand p53 mutation fragments of 168 and 195 bp, respectively, were separated and analyzed. A four-fold decrease in background signals produced from nicked fully matched DNA fragments upon LDR treatment was achieved.

References

[1] M. Khanna, etal, Oncogene, 1999, 18, 27-38

[2] H. Pincas, etal., Nucleic Acids Research, 2004, 32(19), e148