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
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 polymerize 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,
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.