Oligonucleotide Arrays from Aldehyde-Bearing Glass with Coated Background

Abstract

Presented here is a method for preparing small DNA arrays on aldehyde-bearing glass slides. Immobilization involves reductive amination and employs oligonucleotides with 3¢-terminal lysine residues, obtained in high yield from solid phase syntheses. Spot patterns are produced by protecting selected areas of the aldehyde slides with wax, coating the free surface with a methyl triethylene glycol derivative, and removing the wax with dichloromethane. The DNA arrays give better signal to noise ratios in hybridization experiments than slides without passified background.

ChipCheckA Program Predicting Total Hybridization Equilibria for DNA Binding to Small Oligonucleotide Microarrays

“…Once these hot spots for false positives are identified, they may be eliminated by using other probes or, where appropriate, through chemical modifications. 40 As a very first example of a simulation of experimental hybridization results, we have performed a ChipCheck calculation for the microarray data presented in Figure 2 of ref 40. For this, HyTher thermodynamic values with linear correction for microchips were used for the unmodified strands, and the ∆∆H and ∆∆S values for the chl modification reported in Table 4 of ref 38 added to the ∆H and ∆S values of the unmodified, perfectly matched duplex to obtain values for the modified probe.…”

“…38,39 Further, we have taken the molecular cap approach to custom-made oligonucleotide arrays. 40 When performing hybridization experiments in solution, we noticed that mismatches at the termini of short duplexes (which are more sensitive to mismatches than longer duplexes) induced melting point depressions as small as 1.9°C. 38 In fact, there are reports of duplexes with mismatches that are more stable than their fully complementary counterparts.…”

“…For the hybridization buffer, conditions close to those favored by us 40 were entered. A total number of 10 12 probe molecules of a given sequence was chosen, since, with the microarrays currently employed by us, 40 this number per spot was realistic, based on the probe density reported in other work with immobilized oligonucleotides, where high coverage and hybridization efficiencies had been achieved. 42 Numbers of probe strands that are typical for high-density microarrays (spot size approximately 50 µm) may, of course, also be entered when submitting data sets for calculations with ChipCheck.…”

A Direct Glimpse of Cross‐Hybridization: Background‐Passified Microarrays That Allow Mass‐Spectrometric Detection of Captured Oligonucleotides

“…Our initial attempts to detect directly from gold surfaces were unsuccessful, [13] but the refined approach reported herein allows the direct mass spectrometric detection of cross-hybridization. The approach uses background-passified surfaces [14] to prevent nonspecific adsorption of DNA on gold surfaces. [15] Our method involves the capture of DNA strands on the surface by hybridization to immobilized probes, a washing step to remove unbound strands, the addition of the matrix 2,4,6-trihydroxyacetophenone in an ethanolic solution to favor dissociation of duplexes and desorption, and massspectrometric detection of the captured target strands.…”

“…The probe on each spot was complementary to the motif indicated above the spectra, with positions of the variable bases indicated. a) Hybridization experiment with all DNA strands (11)(12)(13)(14)(15)(16)(17)(18)(19), including the perfectly matched (PM) targets. b) Hybridization experiments with DNA strands 12, 13, 14, 16, 17, and 19, that is, without the PM targets.…”