Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.
BACKGROUND
Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base) unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses.


RESULTS
Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface.


CONCLUSIONS
These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.
Cook
Michael A
MA
Centre for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Chan
Chi-Kin
CK
Jorgensen
Paul
P
Ketela
Troy
T
So
Daniel
D
Tyers
Mike
M
Ho
Chi-Yip
CY
D015342:DNA Probes; D001330:Electronic Data Processing / Q000191:economics; D005800:Genes, Fungal; D016681:Genome, Fungal; D018625:Microscopy, Atomic Force; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods* / Q000592:standards; D012441:Saccharomyces cerevisiae
D015342:DNA Probes