SUNY at Albany
June 19-23, 2001
Two linked homopyrimidine PNA oligomers (bis-PNA) invade the DNA double helix forming DNA/(PNA)2 triplexes with one of DNA strands and displacing the other DNA strand. As a result, the displaced strand can hybridize with a DNA probe forming the PD-loop (1). Thus formed DNA-PNA nanostructure can be employed as an artificial primosome. Indeed, we have demonstrated that the participating in the PD-loop oligonucleotide can be used as a primer for the primer-extension reaction by an appropriate DNA polymerase, which is able to extend it via strand displacement. This capability of PD-loop leads to several promising applications.
In particular, we developed a non-denaturing method to sequence linear double-stranded DNA (dsDNA). The method is based on the Sanger dideoxy sequencing protocol, but in our case DNA can be isothermally sequenced without strand separation starting from the PD-loop. As an example, several hundred nucleotides were directly sequenced within a 50-kb-long dsDNA template. The new method promises to perform sequencing on the excessive background of unrelated DNA, thereby evading procedures of target purification (2).
Besides, we have developed a new approach for target quantification directly within duplex DNA. The approach takes advantage of a selective hybridization of oligonucleotide probe to dsDNA, which is locally opened by a pair of bis-PNA oligomers. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase in the presence of single-strand binding (SSB) protein. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis (3).
Other possible applications of the artificial primosome will also be discussed (4).References and Footnotes
Irina V. Smolina, Natalia E. Broude, Heiko Kuhn, Maxim D. Frank-Kamenetskii and Vadim V. Demidov
Center for Advanced Biotechnology, Boston University, 36 Cummington St., Boston, MA 02215, USA email: firstname.lastname@example.org