'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application.
In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.
Stürzl
M
M
Max-Planck-Institut für Biochemie, Department of Virus Research, Martinsried, Germany.
Oskoui
K B
KB
Roth
W K
WK
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D015342:DNA Probes / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000378:metabolism; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives*; D004587:Electrophoresis, Agar Gel; D029822:Enterovirus B, Human / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000235:genetics; D011108:Polymers; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020574:Proto-Oncogene Proteins c-sis; D012313:RNA / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D000199:Actins; D015342:DNA Probes; D004277:DNA, Single-Stranded; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D010982:Platelet-Derived Growth Factor; D011108:Polymers; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; C070202:digoxigenin-11-deoxyuridine triphosphate; D012313:RNA; D004076:Digoxigenin