Title

Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations

Document Type

Article

Publication Date

3-1-2003

Abstract

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 allelespecific 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.

Publication Source (Journal or Book title)

Analytical Chemistry

First Page

1130

Last Page

1140

This document is currently not available here.

Share

COinS