Unrestricted hybridization of oligonucleotides to structure-free DNA |
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Authors: | Gamper Howard B Arar Khalil Gewirtz Alan Hou Ya-Ming |
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Institution: | Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. howard.gamper@jefferson.edu |
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Abstract: | The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays. |
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