An Easy Method to Check the Efficiency of Biotin Endlabelling of Dnafragments
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An easy method to check the efficiency of biotin endlabelling of DNA-fragments
Holmstrom, K., Rasmussen, O.F.
رسالہ:
Nucleic Acids Research
DOI:
10.1093/nar/18.15.4632
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4632 Nucleic Acids Research, Vol. 18, No. 15 An easy method to check the efficiency of biotin endlabelling of DNA-fragments K.Holmstr0m and O.F.Rasmussen Genetic Engineering Group, Lundtoftevej 100 Building 227, 2800 Lyngby, Denmark Submitted May 4, 1990 B Figure 1. Lane 1: Phage Lambda ffimflll-digest. Lane 2: pUC18 BamHI-digest. Lane 3 - 7 : pUC18 BamHI-digests incubated for 4, 8, 12, 16 and 20 minutes, respectively, with T4-DNA-polymerase prior to addition of dATP, dCTP, dGTP and biotin-11-dUTP. Lane 8: pUC18 BamHI-digest incubated for 20 minutes with T4-DNA-polymerase prior to addition of dATP, dCTP, dGTP and dTTP. Lane 9: Phage Lambda HindlU-digest. A: Photograph of 1.5% agarose gel after staining with EtBr. B: Visualization of biotinylated DNA-fragments blotted onto Hybond-N nylon- membrane. while biotin-7-dATP (Bethesda Research Laboratories, USA) seems to be a very poor substrate for T4-DNA-polymerase. However, it seems that the Klenow fragment incorporates biotin-7-dATP at a much higher incorporation rate than does T4-DN A-polymerase. REFERENCE 1. Sambrook.J., Fritsch.E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Downloaded from http://nar.oxfordjournals.org/ at University of Arizona on June 2, 2014 One drawback when using non-radioactive labelling has been that there is no easy test for the degree of incorporation of nucleotide analogues. Here we show that biotinylated DNA-fragments have a lower mobility than non-biotinylated fragments during agarose gel electrophoresis, and that this gives a good indication of the degree of end-labelling of DNA-fragments. This feature can be exemplified as follows: The 2.7 kbp pUC18-vector was restricted with BamHl and exposed to the exonuclease activity of T4-DNA-polymerase for 4, 8, 12, 16 or 20 minutes (1). Subsequently dATP, dCTP, dGTP and biotin-11-dUTP (Sigma, England) was added to concentrations of 200 fiM and 37.5 jtM respectively, and the reactions were allowed to polyme; rize for 30 minutes before they were stopped with 25 mM EDTA. The samples were loaded on a 1.5% agarose-gel and separated by electrophoresis (in TAE-buffer (1)) for 1—2 hours at 5 V/cm. The gel was stained with EtBr and exposed to UV-light to visualize the DNA-bands (figure la). In order to show that the DNA-fragments were indeed biotinylated, the gel was blotted onto Hybond-N nylon-membrane (Amersham Ltd., England) and biotinylated DNA-fragments were visualized with reagents for detection of biotinylated DNA-probes (Dakopatts, Denmark). The filter was allowed to stain for 5 minutes with BCIP/NBT and was then stopped by addition of TE-buffer (figure lb). Figure la shows that the mobility shift is distinct after only 4 minutes of exonuclease activity, and that the mobility decreases further with increasing periods of exonuclease activity due to a higher incorporation of biotin-11-dUTP. This is reflected in figure lb, where an increasing amount of incorporated biotin-residues is seen as an increase in the biotin signal intensity. Another observation is the generation of 'fuzzy' DNA-bands with increasing periods of exonuclease activity. This feature is presumably caused by the lack of synchronisation of the exonuclease activity reactions. The relative change in mobility is dependent on the length of the DNA-fragments investigated. For short (less than 2 — 3 kbp) DNA-fragments the mobility shift will be very distinct, but the mobility difference will be less obvious with increasing size of the fragments. This problem can be circumvented by restricting the labelled DNA-fragments with a restriction enzyme that creates subfragments with an appropriate size for observing the shift in mobility. A good indication of quantitative end-labelling is the absence of DNA-fragments from the labelling reaction with mobility equal to the unlabelled DNA-fragments. We have found that T4-DNA-polymerase incorporates biotin-11-dUTP (Sigma and Enzo Diagnostics Inc., USA) and biotin-16-dUTP (Enzo Diagnostics Inc., USA) equally efficiently,
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