Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations.
Low-density arrays were assembled into microfluidic channels hot-embossed in poly(methyl methacrylate) (PMMA) to allow the detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. Following spotting, the chip was assembled with a cover plate and the array accessed using microfluidics in order to enhance the kinetics associated with hybridization. The array was configured with zip code sequences (24-mers) that were complementary to sequences present on the target. The hybridization targets were generated using an allele-specific ligase detection reaction (LDR), in which two primers (discriminating primer that carriers the complement base to the mutation being interrogated and a common primer) that flank the point mutation and were ligated joined together) only when the particular mutation was present in the genomic DNA. The discriminating primer contained on its 5'-end the zip code complement (directs the LDR product to the appropriate site of the array), and the common primer carried on its 3' end a fluorescent dye (near-IR dye IRD-800). The coupling chemistry (5'-amine-containing oligonucleotide tethered to PMMA surface) was optimized to maximize the loading level of the zip code oligonucleotide, improve hybridization sensitivity (detection of low-abundant mutant DNAs in high copy numbers of normal sequences), and increase the stability of the linkage chemistry to permit re-interrogation of the array. It was found that microfluidic addressing of the array reduced the hybridization time from 3 h for a conventional array to less than 1 min. In addition, the coupling chemistry allowed reuse of the array > 12 times before noticing significant loss of hybridization signal. The array was used to detect a point mutation in a K-ras oncogene at a level of 1 mutant DNA in 10,000 wild-type sequences.
Wang
Yun
Y
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, USA.
Vaidya
Bikas
B
Farquar
Hannah D
HD
Stryjewski
Wieslaw
W
Hammer
Robert P
RP
McCarley
Robin L
RL
Soper
Steven A
SA
Cheng
Yu-Wei
YW
Barany
Francis
F
D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D017931:DNA Primers; D007202:Indicators and Reagents; D008838:Microcomputers; D009154:Mutation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D019904:Polymethyl Methacrylate; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D007202:Indicators and Reagents; D004247:DNA; D019904:Polymethyl Methacrylate