Modifications to Polymerase Chain Reaction Protocols to Enhance Amplification of a G-C Rich Intergenic Region of Herpes Simplex Virus Type-1

Abstract

Herpes simplex virus type-1 (HSV-1) is under active investigation as a potential viral vector for gene therapy. Modifications to the virus are necessary to make HSV-1 safe for potential therapeutic use in humans. The genome of herpes simplex virus type-1 is extremely rich in GC content (>68%), presenting significant challenges from a technical standpoint. The additional hydrogen bonds presented by increased GC base pairs makes DNA strand separation and polymerase chain reaction amplification more difficult. Various DNA polymerases are commercially available, but their results can be variable. We modified the protocols of a commercially available G-C Rich Polymerase System to enhance its efficacy by adding extra steps to improve dissociation of strands of herpes virus DNA and to improve annealing of primers prior to performing PCR. We describe here the protocol modifications that improve the efficacy of amplification of GC-rich sequences of herpes virus DNA. These modifications will help researchers reduce laboratory costs and time associated with working with forms of DNA that are particularly high in GC content. Improving efficacy of HSV-1 polymerase chain reaction will make production of rationally-designed genetically-engineered forms of the virus more technically feasible.