Oligonucleotide Fingerprinting of Arrayed Genomic DNA Sequences Using LNA-Modified Hybridization Probes

Abstract

Recently, we established a robust method for the detection of hybridization events using a DNA microarray deposited on a nanoporous membrane. Here, in a follow-up study, we demonstrate the performance of this approach on a larger set of LNA-modified oligoprobes and genomic DNA sequences. Twenty-six different LNA-modified 7-mer oligoprobes were hybridized to a set of 66 randomly selected human genomic DNA clones spotted on a nanoporous membrane slide. Subsequently, assay sensitivity analysis was performed using receiver operating characteristic (ROC) curves. Comparison of LNA-modified heptamers and DNA heptamers revealed that the LNA modification clearly improved sensitivity and specificity of hybridization experiment. Clustering analysis was applied in order to test practical performance of hybridization experiments with LNA-modified oligoprobes in recognizing similarity of genomic DNA sequences. Comparing the results with the theoretical sequence clusters, we conclude that the application of LNA-modified oligoprobes allows for reliable clustering of DNA sequences which reflects the underlying sequence homology. Our results show that LNA-modified oligoprobes can be used effectively to unravel sequence similarity of DNA sequences and thus, to characterize the content of unknown DNA libraries.

Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain

“…Nucleotides such as RNA, methylated bases, and LNA improve the stability of their hybrid with DNA ( 20 , 26 ) and thus increase <τ disp >: it increases by ∼4 for a 11 nt RNA oligonucleotide (Figure 3A ) when compared to the same DNA oligonucleotide; a single LNA base increases <τ disp > by ∼2, and three LNA bases by ∼10 (Figure 3B ); a single methylated cytosine may increase <τ disp > by ∼1.3 (data not shown).…”

Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain

“…Nucleotides such as RNA, methylated bases and LNA improve the stability of their hybrid with DNA 19,20 and thus increase <τ disp > : it increases by ∼4 for a 11 nt RNA oligonucleotide (Fig.3A) when compared to the same DNA oligonucleotide; a single LNA base increases <τ disp > by ∼2, and three LNA bases by ∼10 (Fig.3B); a single methylated cytosine may increase <τ disp > by ∼ 1.3 (data not shown).…”

“…The displacement time <disp> is almost independent of the salt concentration (see Supp. Nucleotides such as RNA, methylated bases and LNA improve the stability of their hybrid with DNA 19,20 and thus increase <disp> : it increases by ~4 for a 11 nt RNA oligonucleotide (Fig. 3A) when compared to the same DNA oligonucleotide; a single LNA base increases <disp> by ~2, and three LNA bases by ~10 (Fig.…”

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 ].…”