Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations.
Identifieur interne : 000182 ( Ncbi/Curation ); précédent : 000181; suivant : 000183Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations.
Auteurs : Yun Wang [États-Unis] ; Bikas Vaidya ; Hannah D. Farquar ; Wieslaw Stryjewski ; Robert P. Hammer ; Robin L. Mccarley ; Steven A. Soper ; Yu-Wei Cheng ; Francis BaranySource :
- Analytical chemistry [ 0003-2700 ] ; 2003.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : DNA.
- chemical , genetics : DNA.
- chemical : DNA Primers, Indicators and Reagents, Polymethyl Methacrylate.
- genetics : Mutation.
- Microcomputers, Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction.
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 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.
DOI: 10.1021/ac020683w
PubMed: 12641233
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<term>Microcomputers</term>
<term>Mutation (genetics)</term>
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<front><div type="abstract" xml:lang="en">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.</div>
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