Improved PCR by the Use of Disruptors, a New Class of Oligonucleotide Reagents.

Abstract

As a powerful tool, polymerase chain reaction (PCR) has been indispensable and widely used in a large array of applications. In practice, many factors may affect the overall performance of a PCR. One such factor is the stability of intramolecular secondary structure formed within single-stranded template. The higher the stability of such a structure, the more likely it will have adverse effects on PCR performance. Traditionally, chemical reagents believed to reduce the stability of nucleic acid secondary structures, such as DMSO and betaine, have been used to mitigate their adverse effects on PCR performance. However, these reagents have apparent downsides including increasing replication error rate, inhibiting polymerase activity, and being ineffective against secondary structures of very high stabilities. Disruptors, a new class of oligonucleotide reagents, do not exhibit such downsides. They are specifically designed to target intramolecular secondary structures only without any effect on the replication of other regions of the template. Their effective concentration range for improving PCR performance is well tolerated by PCR. And they are very effective in improving PCR performance on templates that are notoriously difficult to amplify by PCR even in the presence of DMSO or betaine, e.g., the inverted terminal repeat of adeno-associated virus (AAV-ITR). In this chapter, the application of disruptors in PCR is described with AAV-ITR as the example template.