A Method of Using Heavy Mineral Oil for Performing "Hot-Start" Amplification of Rare Nucleic Acids

3′-Protected 2′-Deoxynucleoside 5′-Triphosphates as a Tool for Heat-Triggered Activation of Polymerase Chain Reaction

“…When one 3′-THF dNTP was substituted for the respective dNTP in PCR mixture, a substantial improvement in amplification of the 365-bp amplicon was evident, with 3′-THF dATP and 3′-THF dTTP derivatives showing the best performance. The effect of the combined action of two, three, or four 3′-THF derivatives was much stronger, resulting in a drastic reduction of primer dimers and improved performance and specificity of PCR (lanes [10][11][12][13][14][15][16][17][18][19][20]. At this time, we cannot explain the apparent and reproducible differences in the efficiency of the suppression of primer dimers observed for 3′-THF derivatives of dCTP, dTTP, dATP, dGTP, and their combinations.…”

“…Hot start PCR activation can be also achieved by placing a temporary physical barrier, such as a wax or sequester, between critical PCR components. 6, [16][17][18][19] The barrier melts or dissolves at high temperature of the PCR cycling, allowing the critical components to mix with the rest of the solution, resulting in improved PCR performance. However, these technical solutions are laborious and prone to mechanical errors, especially for high-throughput applications.…”

“…The original hot start PCR approach is performed by a temporary withdrawal of critical component(s) of the PCR mixture, such as DNA polymerase, primers, or magnesium ion from the solution, thus preventing the primer extension reaction from happening. , The critical PCR component is added to the tube only after the whole mixture reaches high stringency conditions during the initial heat denaturing step. Hot start PCR activation can be also achieved by placing a temporary physical barrier, such as a wax or sequester, between critical PCR components. ,− The barrier melts or dissolves at high temperature of the PCR cycling, allowing the critical components to mix with the rest of the solution, resulting in improved PCR performance. However, these technical solutions are laborious and prone to mechanical errors, especially for high-throughput applications.…”

Heat‐Activatable Primers for Hot‐Start PCR: Oligonucleotide Synthesis and Basic PCR Setup

“…The first design of the hot-start PCR approach relied on a temporary withdrawal of one or two critical components of the PCR mixture, such as DNA polymerase, primers, or magnesium ion, from the solution, thus preventing undesired primer extension reactions from occurring (D'Aquila et al, 1991;Kaijalainen et al, 1993;Riol et al, 1994;Horton et al, 1994;Barnes and Rowlyk, 2002). The critical component(s) was added only after the PCR mixture reaches a high stringency conditions during the initial heatdenaturing step.…”

PCR and Its Variations

“…This barrier can be created by adding wax over an incomplete PCR reaction mixture in a tube (Bassam and Caetano-Anolles, 1993;Horton et al, 1994;Riol et al, 1994). The wax can be preformulated for PCR reactions or can be in bulk form, such as paraffin.…”