Template-directed fluorogenic oligonucleotide ligation using "click" chemistry: detection of single nucleotide...

A novel nonfluorescent alkyne-modified coumarin phosphoramidite was synthesized and successfully incorporated into oligonucleotides, which were then used in highly efficient DNA interstrand cross-linking and ligation reactions via "click" chemistry. The template-directed fluorogenic ligation "click" chemistry reaction was used for single nucleotide polymorphism analysis, where the target DNA catalyzes the ligation of two nonfluorescent probes to generate a fluorescent product. The upstream oligonucleotide probe is a nonfluorescent alkyne-modified coumarin and the downstream probe is an azide-modified oligonucleotide. When bound to a fully complementary template, the oligonucleotides ligated to produce a fluorescent product with a fluorophore at the ligation point. Wild-type and mutant p53 alleles were used to demonstrate that template-directed fluorogenic oligonucleotide ligation is sequence-specific and is capable of single nucleotide discrimination under mild conditions, even without the removal of unreacted probes.