Ion-Exchange Capture of Labeled Pyrrolidinyl Peptide Nucleic Acids for DNA Sequence Determination

Peptide nucleic acid (PNA) is a new class of DNA mimic, which is potentially useful for diagnostic and therapeutic applications due to its high affinity and specificity of binding to target nucleic acids. The goal of this work is to develop a simple and sensitive fluorimetric-based detection of DNA sequences using new fluorescent labeled PNA probes previously developed in our laboratory. The method relies on the preferential absorption of PNA.DNA hybrid (negatively charged) over unhybridized PNA (neutral) on an anion exchange solid support. This enables detection of the PNA-nucleic acid interactions, which will take place only when the PNA and DNA sequences are fully complementary directly on the solid support by fluorescence microscopy. With the high specificity of this new PNA-based technology, single mismatches in 9-13 bases target DNA sequences were readily distinguished. Based on this principle, a prototype for detection of single nucleotide polymorphisms (SNP) without the need for labeling the DNA substrates has been developed.