Open AccessSeparation of complementry strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy
Author(s) -
Hajo Delius,
Harm van Heerikhuizen,
Jill Clarke,
Barbara Koller
Publication year - 1985
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/13.15.5457
Subject(s) - biotinylation , avidin , biology , biotin , agarose , streptavidin , dna , hpaii , microbiology and biotechnology , biochemistry , gene expression , gene , dna methylation
A method for the separation of complementary strands with the help of the biotin-avidin system is described. Restriction fragments were terminally labeled at both ends with biotinylated nucleotides. The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column. The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide. Contamination of the separated strands by complementary single strands was less than 4%.-Separated linear single strands of the vector pEMBL were prepared. On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences. -The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.