Reducing Nonspecific Amplification
PCR is a widely accepted thermal cycling process that provides approximately 106-fold amplification of a nucleic acid target of interest. Dr. Paul said that it is increasingly used in high-stringency applications such as molecular diagnostics where the requirement is to detect down to as few copies as possible without false positives or false negatives.
“The process can be plagued by competing off-target amplicon formation such as mispriming and primer dimer formation,” she noted. TriLink’s Hot Start activation is a strategy to reduce nonspecific amplification during the less stringent set-up stages of PCR, she explained.
The goal of Hot Start technologies is to block primer extension at lower temperatures, but to allow for reactivation of the process at higher temperatures. TriLink has developed two approaches to Hot Start activation in PCR that employ chemical modifications to two widely underutilized components of the reaction—the primers and the dNTPs.
The modified primer technology employs a thermolabile blocker, in the form of 4-oxo-tetradecyl, to the 3´ phosphodiester backbone of the primer molecule. The blocker is released at 94ºC, leaving a normal, unmodified primer that can be extended. The CleanAmp™ Turbo Primer features one blocker moiety while the Precision Primer features a double substitution of the group.
CleanAmp Turbo Primers are used in fast cycling (two-step protocols) for multiplexed PCR of DNA templates. CleanAmp Precision Primers are applied in standard cycling (three-step protocols) and in one-step reverse transcription PCR.
Dr. Paul noted that this modified primer technology is of particular utility in one step RT-PCR set-ups, where the lower temperature reverse-transcription step is accomplished with a poly-dT reverse transcription primer and also includes PCR primers. These primers are blocked from extension by CleanAmp Precision modifications until the tube is heated up to PCR temperatures.
According to Dr. Paul, CleanAmp Precision PCR Primers enhance results in multiplex one-step RT-PCR when compared to unmodified PCR primers, which are generally available throughout the reaction. Dr. Paul said that results from tests in liver RNA indicate that the CleanAmp Precision Primer modifications show promise for relative RNA quantification in real-time one-step RT-PCR.
Hot Start activation works in largely the same way with TriLink’s modified deoxyribonucleotide triphosphate (dNTP) technology. The thermolabile protective group in this case is a tetrahydrofuranyl modification to the 3´ hydroxyl, which has been shown to result in improved PCR performance for several targets.
In a demonstration of increased sensitivity, the use of CleanAmp dNTPs detected DNA at 5,000, 500, 50, and 5 copy levels, unmodified dNTPs were effective at only the 5,000 and 500 copy levels. CleanAmp dNTPs were also found to enhance PCR specificity in comparison with unmodified dNTPs when used in combination with CleanAmp Precision Primers, Dr. Paul said, essentially eliminating detectable primer-dimer formation, indicating that the use of CleanAmp dNTP and CleanAmp primers together provide a synergistic effect.