LNA-Modified Oligodeoxynucleotide Hybridization with DNA Microarrays Printed on Nanoporous Membrane Slides

Abstract

Abstract:We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to ~4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more.

In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification

“…The synthesis of Locked Nucleic Acid (LNA) [ 6 ] overcame these limitations as LNA modified nucleotides confer low cytotoxicity, high thermostability, resistance to nucleases and stable hybridization abilities with target sequences [ 7 ]. Enhanced nucleic acid recognition by LNA-containing oligonucleotides made them desirable for many applications in molecular biology, including genotyping [ 8 ] or single nucleotide polymorphism (SNP) analysis [ 9 ], hybridization [ 10 , 11 ], decoy and fluorescence polarization [ 12 ], expression profiling or microarray [ 13 ], allele-specific PCR [ 14 ], fluorescent in situ hybridization (FISH) analysis [ 15 ], alteration of intron splicing and LNAzymes [ 16 ], 5′-nuclease assay [ 17 ], real-time PCR [ 18 ], siRNA [ 19 ], microRNA [ 20 ] and antisense [ 21 ].…”