Temperature-assisted cyclic hybridization (TACH): an improved method for supercoiled DNA hybridization.
Accurate hybridization is dependent on the ratio between sequence-specific and unspecific binding. Dissociation of unspecifically bound, while maintaining specifically hybridized, nucleic acids are key steps to obtain a well-defined complex. We have developed a new method, temperature-assisted, cyclic hybridization (TACH), which increases cognate binding at the expense of unspecific hybridization. The method was used for optimizing binding of peptide nucleic acid (PNA) to supercoiled plasmids and has several advantages over previous methods: (1) it reduces the required amount of bis-PNA by three- to fourfold; (2) it results in less unspecific binding; (3) it extends cooperative hybridization, from 3 bp to 5 bp between two adjacent binding sites; and (4) it decreases the aggregation of bis-PNA. This method might be extended to other forms of hybridizations including the use of additional nucleic acids analogs, such as locked nucleic acid (LNA) and, also, to other areas where PNAs are used such as fluorescence in situ hybridization (FISH), microarrays, or in vivo plasmid delivery.
Oprea
Iulian I
II
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Huddinge, Sweden. iulian.oprea@ki.se
Simonson
Oscar E
OE
Moreno
Pedro M D
PM
Viola
Joana R
JR
Lundin
Karin E
KE
Smith
C I Edvard
CI
D000704:Analysis of Variance; D000818:Animals; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature
D004278:DNA, Superhelical; D020135:Peptide Nucleic Acids