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Detection of single nucleotide polymorphisms through the application of lifetime fluorescent probes
Author(s) -
Karlstad Jordan P.,
Gonnella Thomas P.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a526-b
Subject(s) - fluorophore , single nucleotide polymorphism , snp genotyping , genotyping , oligonucleotide , snp , fluorescence , genetics , biology , microbiology and biotechnology , oligomer restriction , gene , genotype , physics , optics
Fluorescence‐based approaches for genotyping single nucleotide polymorphisms (SNPs) commonly involve enzymatic cleavage, stringent control of hybridization temperature, or extensive post amplification clean up. Our research to SNP genotyping employs fluorescence lifetime probes, which consist of a single fluorophore that is covalently bound to a oligonucleotide complementary to a SNP allele. We require that the matched and mismatched forms correspond to different lifetimes of the fluorophore. In other words, fluorophore lifetime reports whether the base has entered into the double strand or is mismatched. The constraints on hybridization conditions are greatly relaxed and enzymatic cleavage is not required. This fluorescent lifetime‐based technology allows resolving SNP alleles in both the homozygous and heterozygous states using a single lifetime probe. Initial studies have been conducted on the human β‐globin gene, known to cause sickle cell anemia. Our results show the fluorophore lifetime is measurably different for the mutant and wild forms. We are currently characterizing the effects of probe/target base pair mismatch beyond the initial SNP. Preliminary studies suggest fluorescence polarization data likely provides additional specificity. This research was supported by the North Dakota INBRE grant.