Improved PCR specificity with Hot Start PCR primers

The polymerase chain reaction (PCR) is an indispensable DNA amplification technique that has been exploited in numerous areas, including molecular diagnostics. One major drawback of PCR is the competing amplification of undesired off-target products, which primarily occurs at ambient temperatures prior to PCR cycling (1). Hot Start PCR techniques aim to block the extension of primers in nonspecific complexes until higher, more stringent temperatures, thereby reducing off-target amplification. Herein, we describe a primer-based approach to Hot Start activation. CleanAmpTM Primers contain temperaturesensitive 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. This generates the unmodified primer, which can now be a substrate for the DNA polymerase. As a consequence, the primers are extended under higher-stringency conditions, diminishing the chance of amplifying off-target products. There are two types of CleanAmpTM Primers, differing in the number of OXT groups that they contain. CleanAmpTM Turbo Primers contain only a single OXT modification at the 3′-terminal internucleotide linkage, while CleanAmpTM Precision Primers contain OXT modifications at the two 3′-terminal internucleotide 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). CleanAmpTM 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. CleanAmpTM 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.