Molecular hybridization probes prepared with 4-aminooxybutylamine.
A versatile method is described for preparing nonradioactive DNA probes for molecular hybridization. The method is based on the transamination reaction of double-stranded DNA with 4-aminooxybutylamine (ABA). To optimize the procedure for obtaining stable and sensitive hybridization probes, time of modification, pH, and reaction temperature were varied. The optimal reaction conditions allowed the preparation of nonradioactive-labeled DNA probes that met demands for optimal length, modification degree, stability, and sensitivity. The use of 4-aminooxybutylamine as a bifunctional reagent for DNA modification allowed the possibility of choosing an appropriate reporter group: biotin or one of the fluorochromes. For probes carrying biotin, a high specificity and high sensitivity detection limit of 1 pg of target DNA were demonstrated. In addition, the applicability of probes carrying fluorochromes for multicolor direct fluorescence in situ hybridization was described.
Adarichev
V A
VA
Institute of Cytology and Genetics SD RAS, 10 Lavrentyeva Avenue, Novosibirsk 630090, Russia.
Kalachikov
S M
SM
Kiseliova
A V
AV
Dymshits
G M
GM
D000818:Animals; D001710:Biotin / Q000737:chemistry; D002082:Butylamines / Q000737:chemistry*; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007202:Indicators and Reagents; D051379:Mice; D008907:Mink
C116451:4-aminooxybutylamine; D002082:Butylamines; D015342:DNA Probes; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D001710:Biotin; D004247:DNA; D019793:Fluorescein