Peptide nucleic acids are molecules in which the bases are conjugated to a polyamide back-bone. The distance between bases and the complementarity are the same as in a DNA or RNA molecule.
The PNA probe is hybridized to a DNA sample amplified by PCR, denatured at low ionic strength, and resolved by CE. The neutral backbone of PNA ensures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA.
DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands.
This method can be used to perform multiplex analyses on several mutations simultaneously when using different amplicon lengths and a set of mutation-specific PNAs. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is further characterized by its interaction with a specific PNA that can differentiate between wild-type and mutant alleles.
This approach has been applied to the detection of R553X and R1162X single-base mutations in patients with cystic fibrosis and to identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene.
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