Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations.
We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90 degrees to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be approximately 41pmolcm(-2), near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.
Situma
Catherine
C
Center for Biomodular Multi-Scale Systems, Louisiana State University, Baton Rouge, LA 70803, USA.
Wang
Yun
Y
Hupert
Mateusz
M
Barany
Francis
F
McCarley
Robin L
RL
Soper
Steven A
SA
D000327:Adsorption; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D007553:Isotope Labeling; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D015345:Oligonucleotide Probes; D017354:Point Mutation / Q000235:genetics*; D019904:Polymethyl Methacrylate / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays
D015345:Oligonucleotide Probes; D019904:Polymethyl Methacrylate