Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.
A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA. Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.
D001710:Biotin; D003124:Colorimetry; D004247:DNA / Q000032:analysis*; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D005374:Filtration; D009693:Nucleic Acid Hybridization; D009757:Nylons
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D009757:Nylons; C024107:2'-deoxycytidine 5'-triphosphate; D001710:Biotin; D004247:DNA