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

Abstract

2'-Deoxyribonucleoside-3'-O-(4-oxotetradec-1-yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phosphotriester protecting groups at 3'-ultimate and penultimate internucleotide linkages. The OXT-modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT-modified oligonucleotide primers and for performing hot-start PCR are described.

Improved PCR specificity with Hot Start PCR primers

“…They were prepared using fast deprotecting phosphoramidites (Glen Research, Sterling, VA, USA) with OXT modifications introduced using CleanAmp Amidites (TriLink BioTechnologies, Inc. or Glen Research). After synthesis, CleanAmp Primers were isolated and purified using a solid phase extraction step (2) as described in further detail at trilinkbiotech.com/cleanamp/amidites.…”

“…Herein, we describe a primer-based approach to Hot Start activation. CleanAmp Primers contain temperature-sensitive 4-oxo-tetradecyl (OXT) phosphotriester modifications which can be introduced at the 3-terminal phosphodiester linkages of any primer (2,3). The OXT group blocks DNA polymerase primer extension at low-stringency conditions, but dissociates at the higher temperatures of PCR.…”

“…CleanAmp Turbo Primers contain only a single OXT modification at the 3-terminal internucleotide linkage, while CleanAmp Precision Primers contain OXT modifications at the two 3-terminal inter-nucleotide linkages. The OXT modifications, which can be introduced using standard solid phase oligonucleotide synthesis, are removable at elevated temperatures to produce the corresponding unmodified primer (2,4). CleanAmp Turbo Primers are best suited for applications in which the primers need to be more readily available, such as fast cycling PCR and multiplex PCR.…”

“…CleanAmp Turbo Primers are best suited for applications in which the primers need to be more readily available, such as fast cycling PCR and multiplex PCR. CleanAmp Precision Primers contain two modification groups and therefore require a slightly longer initial denaturation time to be activated (2). This slower-releasing characteristic provides an advantage in applications that require lower-temperature incubation, such as the reverse transcription (RT) step in a one-step RT-PCR protocol.…”

Heat‐Activatable Primers for Hot‐Start PCR and Hot‐Start One‐Step RT‐PCR: Endpoint and Real‐Time Experiments

“…Typically, hot-start activation in PCR The protocols in this unit describe a method in which the primers, rather than the polymerase, are activated by increased temperature. This primer-based hot-start PCR approach uses thermolabile phosphotriester primer modifications that can easily be introduced onto the 3 terminal phosphodiester linkages of standard primer sequences via standard oligonucleotide protocols (Lebedev, 2009;Fig. 15.9.1).…”