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

Abstract

Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3' end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymerase suitable for your application is not available.

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

“…Hot Start is one of the well established and convenient methods that reduces nonspecific amplification during the initial stages of PCR and increases PCR amplification with higher sensitivity, specificity and yield. However, it has certain limitations: 1) long initial activation steps, 2) reduction in performance of the DNA polymerase or inadequate specificity control (Ashrafi and Paul, 2009). Gradient and TD-PCR (Don et al, 1991) were also used by researchers as per their region of interest and requirement of study.…”

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

“…The above cycling parameters are recommended as an example for the λ-phage DNA system. For more detailed recommendations on PCR with OXT-modified primers, see Ashrafi and Paul (2009). 5.…”

Improved PCR specificity with Hot Start PCR primers

“…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.…”