Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification

Selective binding of looped oligonucleotides to a single-stranded DNA and its influence on replication in vitro.

Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides

Looped oligonucleotides form stable hybrid complexes with a single-stranded DNA.

Several new branched (1, 2), circular (9) and looped oligonucleotides (14-17) were synthesized. 3'-Deoxypsicothymidine was employed to create the site of branching when required. The circular and looped structures were obtained by oxidative disulfide bond formation between mercaptoalkyl tether groups. All the oligonucleotides prepared contained two T11 sequences, and the branched and looped oligomers an additional alternating CT sequence. The melting experiments revealed that the branched oligonucleotides form relatively weak hybrid (double/triple helix) complexes with the single-stranded oligodeoxyribonucleotide, showing a considerable destabilizing effect produced by the structure at the point of branching. The data obtained with looped oligonucleotides demonstrated considerable stabilization of the hybrid (double/triple helix) complexes with the complement. The data reported may be useful in attempting to design new antisense or antigene oligonucleotides capable of forming selective and stable bimolecular hybrid complexes with nucleic acids