novel endonuclease IV post-PCR genotyping system

• Published in: Nucleic Acids Research 2006-11, Vol.34 (19), p.e128-e128
• Main Authors: Kutyavin, Igor V, Milesi, Dave, Belousov, Yevgeniy, Podyminogin, Mikhail, Vorobiev, Alexei, Gorn, Vladimir, Lukhtanov, Eugeny A, Vermeulen, Nicolaas M.J, Mahoney, Walt
• Format: Article
• Language: English
• Subjects: Agouti Signaling Protein; Alleles; Base Pair Mismatch; Deoxyribonuclease IV (Phage T4-Induced) - metabolism; DNA Breaks, Double-Stranded; Escherichia coli Proteins - metabolism; Fluorescent Dyes - chemistry; Genes, APC; Genotype; Intercellular Signaling Peptides and Proteins - genetics; Methods Online; Nucleic Acid Hybridization - methods; Oligonucleotide Probes - chemical synthesis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Thermodynamics
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkl679

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.