Colorimetric detection of anthrax DNA with a peptide nucleic acid sandwich-hybridization assay

Technologies for genomic detection most commonly use DNA probes to hybridize to target sequences. To achieve required sensitivity, the use of PCR to amplify target sequences has remained standard practice in many labs. Direct detection methods that eliminate the requirement for a PCR step could afford faster and simpler devices that can be used outside of a laboratory. We have developed a sandwich-hybridization DNA detection system in which the DNA detection probes are replaced with a class of synthetic nucleic acid mimics called peptide nucleic acids (PNAs). There are numerous advantages to using PNA instead of DNA probes in hybridization assays, including complete resistance to degradation by enzymes, increased sequence specificity to complementary DNA, and higher stability when bound with complementary DNA. In this communication, several different strategies are described to improve and fine-tune the properties of PNA so that anthrax DNA can be detected at a 10 zmol limit. Key to the success of these strategies is the ability to introduce chemical modifications into the PNA to improve thermal stability and to increase the number of biotin groups to which a horseradish peroxidase-avidin conjugate can bind.