Impact of surface chemistry and blocking strategies on DNA microarrays.
The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.
Taylor
Scott
S
Center for Bioelectronics, Biosensors and Biochips (C3B), Virginia Commonwealth University, PO Box 843038, 601 West Main Street, Richmond, VA 23284-3038, USA.
Smith
Stephanie
S
Windle
Brad
B
Guiseppi-Elie
Anthony
A
D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D050091:Dendrimers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D011073:Polyamines / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011437:Propylamines / Q000737:chemistry; D015203:Reproducibility of Results; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties
C403136:3-glycidoxypropyltrimethoxysilane; D018076:DNA, Complementary; D050091:Dendrimers; D015345:Oligonucleotide Probes; C104700:PAMAM Starburst; D011073:Polyamines; D011437:Propylamines; D012821:Silanes; C088294:3-aminopropyltrimethoxysilane; D011107:Polylysine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases