11.2.1. Nuclease protection assays (RPAs and SNPAs)

Nuclease protection assays are an efficient way to analyse the genetic complexities of natural populations of organisms (Kurath et al., 1993; Arens, 1999; Wang and Chao, 2005). A labelled probe is hybridised to the nucleic acid sample of a population of organisms (usually viruses or other pathogens, sometimes related mRNA species) and then digested with RNAse (RNA probe) or S1-nuclease (DNA probe) which will cut the probe wherever there is a mismatch between probe and target. The resulting pattern of digested probe fragments, revealed by gel electrophoresis, is qualitatively and quantitatively indicative of the mismatch polymorphisms present in the nucleic sample. These procedures are called RNAse Protection Assay (RPA) and S1-Nuclease Protection Assay (SNPA).

Pros   

  • Entire populations can be screened for genetic complexity within the target sequence in a single reaction.
  • The polymorphic sites can be mapped on the genome, through the sizes of the fragments produced.

Cons 

  • Assay is limited to about 300 bases, requiring many assays to cover a genome.
  • Protocols are complex and subject to errors.
  • The nature of the polymorphs requires further analysis.