Separation of complementary strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy.
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.
H
H
Delius
H
H
van Heerikhuizen
J
J
Clarke
B
B
Koller
D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D002021:Buffers; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005055:Euglena / Q000235:genetics; D005293:Ferritins; D019791:Guanidine; D006146:Guanidines; D008854:Microscopy, Electron; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D010047:Ovalbumin / Q000031:analogs & derivatives; D010957:Plasmids; D012313:RNA / Q000235:genetics*; D019809:Streptavidin
D001426:Bacterial Proteins; D002021:Buffers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D006146:Guanidines; D009692:Nucleic Acid Heteroduplexes; D001360:Avidin; D012313:RNA; D001710:Biotin; D010047:Ovalbumin; D004247:DNA; D005293:Ferritins; D019809:Streptavidin; D019791:Guanidine