Journal
BIOCONJUGATE CHEMISTRY
Volume 24, Issue 7, Pages 1226-1234Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bc4001678
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Funding
- UWM Research Growth Initiative [RGI101 x 234]
- Great Milwaukee Foundation
- UWM
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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.
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