DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions.
Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.
Belosludtsev
Y
Y
Genometrix Inc., 3608 Research Forest Drive, Suite B7, The Woodlands, Texas 77381, USA. yurib@genometrix.com
Iverson
B
B
Lemeshko
S
S
Eggers
R
R
Wiese
R
R
Lee
S
S
Powdrill
T
T
Hogan
M
M
D000089:Acetic Anhydrides / Q000378:metabolism; D000327:Adsorption; D020137:Base Pair Mismatch / Q000235:genetics; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D005898:Glass; D018832:Molecular Chaperones / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D055672:Static Electricity; D013379:Substrate Specificity / Q000235:genetics; D013387:Succinic Anhydrides / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry
D000089:Acetic Anhydrides; D015342:DNA Probes; D018832:Molecular Chaperones; D009838:Oligodeoxyribonucleotides; D013387:Succinic Anhydrides; C031800:acetic anhydride; C031801:succinic anhydride