Improved Print and QC Methods for Oligonucleotide Arrays

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We suspect that the hydrazide-modified probes are covalently coupling with the amine-slide surfaceand further that this reaction does not directly involve the primary amine group but rather is an interactionwith other elements of the surface coating. Attachment of hydrazide-oligos varies significantly betweenamine slides from different sources. A total of four different sources were tested (data not shown) and theCorning GAPSII and UltraGAPS surfaces were the most reactive. Increased probe-density and greaterultimate sensitivity can be achieved using IDT’s print buffer and hydrazide-modified probes with CorningGAPSII slides. This combination consistently yields more intense hybridization spot signals with greateruniformity than any other combination tested.A) 3X SSC1.5M Betaine IDT 50% DMSOUltraGAPS no heat couplingB) 3X SSC1.5M Betaine IDT 50% DMSOGAPSII no heat couplingC) 3X SSC1.5M Betaine IDT 50% DMSOSuperAmine no heat coupling1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-h1-u1-a1-h1-A1-H7-u7-a7-hUltraGAPS w/heat couplingGAPSII w/heat couplingSuperAmine w/heat couplingEffect of Attachment Chemistry, Print Buffer, and HeatCoupling on Corning UltraGAPS SurfaceEffect of Attachment Chemistry, Print Buffer, and HeatCoupling on Corning GAPSII SurfaceEffect of Attachment Chemistry, Print Buffer, and HeatCoupling on TeleChem SuperAmine Surfaceno heatheatno heatheatno heatheat100%100%100%80%80%80%60%60%60%40%40%40%20%20%20%0%1u-sb1a-sb1h-sb1A-sb1H-sb7u-sb7a-sb7h-sb1u-IDT1a-IDT1h-IDT1A-IDT1H-IDT7u-IDT7a-IDT7h-IDT1u-D1a-D1h-D1A-D1H-D7u-D7a-D7h-DAttachment Chemistry & Print Buffer0%1u-sb1a-sb1h-sb1A-sb1H-sb7u-sb7a-sb7h-sb1u-IDT1a-IDT1h-IDT1A-IDT1H-IDT7u-IDT7a-IDT7h-IDT1u-D1a-DAttachment Chemistry & Print Buffer1h-D1A-D1H-D7u-D7a-D7h-D0%1u-sb1a-sb1h-sb1A-sb1H-sb7u-sb7a-sb7h-sb1u-IDT1a-IDT1h-IDT1A-IDT1H-IDT7u-IDT7a-IDT7h-IDT1u-D1a-DAttachment Chemistry & Print Buffer1h-D1A-D1H-D7u-D7a-D7h-DFigure 2: Specific Target Hybridizations Comparing Print Buffers and AttachmentChemistries on Different Amine Surfaces. 70mer oligo probes (1 is 25.7% GC and 7 is 55.7% GC) wereprinted @25µM using 3 different print buffers on UltraGAPS (A), GAPSII (B), and SuperAmine (C) slideswith a 3 hour humidity treatment, followed either with (bottom array) or without (top array) heat-coupling.Arrays were hybridized with Cy3-40mer specific-targets @200nM each in 1X HybIt® (TeleChem). Imagesare 5µm resolution scans using ScanArray® 5000 (Perkin Elmer) at 63/63 laser (power/gain) settings. (Pseudocolorscale: black
Comparison of Attachment Chemistry & Print Buffer on3 Commercial Amine Surfaces (w/out heat coupling)UltraGAPS GAPSII SuperAmineRelative % Signal(bkg sub ave Gross DLU/mm2)100%80%60%40%20%0%1u-sb1a-sb1h-sb1A-sb1H-sb7u-sb7a-sb7h-sb1u-IDT1a-IDT1h-IDT1A-IDT1H-IDT7u-IDT7a-IDT7h-IDT1u-D1a-D1h-D1A-D1H-D7u-D7a-D7h-DAttachment Chemistry & Print BufferFigure 3: Hydrazide Attachment Chemistry and IDT’s Print Buffer Provides ImprovedPerformance on the Corning GAPSII Surface.Improved Array QC Reagent and MethodsFluorescent array QC reagents from four sources (Sigma, Genetix, Molecular Probes, andTeleChem) were tested for their ability to visualize oligonucleotide probes printed on CorningUltraGAPS slides. For the range of GC content oligos examined (25% - 60%), none of thesefour reagents reliably identified all the printed probes. We therefore developed our own reagentto QC arrays that would better meet the need for a reagent and method that could be used todetect the widest possible variety of probe sequences. A Cy3-labeled random 9meroligonucleotide was synthesized with base composition adjusted to enhance relativehybridization of AT-rich domains. Hybridization solutions and protocols were developed thatoptimized signal while keeping background low; where possible, similar optimization wasattempted for the other reagents. Performance of the new IDT Cy3-nonamer QC reagent wascompared with the other four commercially available array QC reagents for detection of printedoligos and is shown in Figure 4. Using optimized protocols, the IDT Cy3-nonamer and theTeleChem SeeIt reagent detected all eight of the oligo-probes printed, although the TeleChemreagent needed to be used at 5-fold higher concentration. Improved reagent design andhybridization protocols enable use of the IDT Cy3-nonamer at much lower levels (2µM vs.7.5-10µM) than other reagents, which helps maintain low background further improvingvisualization of spotted oligos with low GC content while reducing cost.The IDT Cy3-nonamer reagent was next tested along with the other four QC products onhigh-density arrays made with PCR products from a cDNA expression library (Figure 5).Interestingly, the new IDT Cy3-nonamer showed similar improved performance whenhybridized against the long PCR product probes as with shorter oligonucleotide probes.
Page 1 and 2: Improved Print and QC Methods for O
Page 3 and 4: Table 1:70mer Oligo-Probe Model Set
Page 5: expected this modification to offer
Page 9 and 10: 2µMIDT Nonamer60 mins10µMTeleChem

Improved Print and QC Methods for Oligonucleotide Arrays

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