© 7994 Oxford University Press
Nucleic Acids Research, 1994, Vol. 22, No. 15
3243-3244
A method for avoiding mis-priming when sequencing with
Dynabeads
Mark G.Thomas*, Kevin W.P.Miller, Charles E.Cook, Jr and Erika Hagelberg
Department of Biological Anthropology, University of Cambridge, Downing Street, Cambridge
CB2 3DZ, UK
Received June 17, 1994; Accepted July 1, 1994
* To whom correspondence should be addressed
protocol (4). Figure 1 shows the results of sequencing part of
the human mitochondrial DNA D-loop region (5) with and
without a stringency wash of the Dynabead/template/sequencing
primer complex. The stringency wash proved effective both in
removing cross-banding due to mis-priming and in increasing
the signal generated from correctly primed template DNA.
Furthermore, this improvement occured over a broad temperature
range (45 to 55°C) and with a minimum number of washes (two).
In addition to the advantage of avoiding mis-priming, this
method overcomes the need for the 1:1 (primer:template) molar
stoichiometry required for effective sequencing (4). The process
takes as little as 5 minutes for 24 samples and thus may be
employed routinely as a precaution against mis-priming. Although
we have not tested this procedure in automated sequencing, it
should prove useful since automated T7 polymerase sequencing
is known to be prone to primer specificity problems (personal
communication, Y.Fosse, Automated DNA Sequencing Service,
King's College, London). This method should also be useful for
sequencing templates amplified with degenerate primers if one
of the primers is used as the sequencing primer, as degenerate
primers will anneal to templates with lower sequence specificity
than non-degenerate primers.
REFERENCES
1. Sanger,F., Niklen,S. and Coulson.A.R. (1977) Proc. NatlAcad. Sci. USA
74, 5463-5467.
2. Pisa-Williamson.D. and Fuller.C.W. (1992) Comments 19, 2 9 - 3 6 . United
Sates Biochemical Corporation, Cleveland, OH.
3. Hultman.T., Stahl,S., Hornes.E. and Uhlen,M. (1989) Nucleic Acids Res.
17, 4937-4946.
4. Tabor,S. and Richardson.C.C. (1987) Proc. Natl Acad. Sci. USA 84,
4767-4771.
5. Anderson.S., Bankier,A.T., Barrell.B.G., de Bruijn,M.H., Coulson,A.R.,
Drouin,J., Eperon,I.C, Nierlich.D.P., Roe,B.A., Sanger.F., Schreier.P.H.,
Smith,A.J., Staden,R. and Young.I.G. (1981) Nature 290, 457-465.
Downloaded from http://nar.oxfordjournals.org/ at University College London on May 20, 2015
Mis-priming of DNA template is one of the most common
problems associated with the dideoxy chain termination method
of DNA sequencing (1). It can be a particular problem with GCrich primers and templates, or with templates containing repeats.
Careful primer design with the use of primer design software
can alleviate many of these problems when sequencing known
sequences but is not possible with unknown sequences. Taq
sequencing is gaining popularity and should not, in theory, suffer
from the problems of mis-priming, but there are many who
believe it has yet to be optimised and who prefer more traditional
protocols. Mis-priming may be partially overcome by the use
of glycerol-tolerant sequencing gels (2), allowing sequencing
extension reactions to be carried out in the presence of high
concentrations of glycerol and thus at higher temperatures. We
have found that a simple stringency wash can eliminate mispriming completely when using the Dynabeads/biotin system
(Dynal Ltd, Merseyside, UK) for the generation of singlestranded DNA template (3).
In detail, the Dynabead-bound single-stranded DNA was
prepared according to Hultman et al. (3). Following a final TE
wash (10 raM Tris-HCl pH 7.5, 1 mM EDTA), the beads were
mixed with Sequenase (U.S. Biochemical, Cleveland, OH, USA)
reaction buffer (final concentration 40 mM Tris-HCl pH 7.5,
50 mM NaCl, 20 mM MgCl2) and 5 ng of sequencing primer.
The mix was heated to 65 °C for 3 minutes and then cooled slowly
to 30°C over a period of 15 minutes. Meanwhile, the Dynabeads
magnetic particle separator (MPC) and 1X Sequenase reaction
buffer were preheated in a water bath set to a few degrees below
the Tm of the sequencing primer. Following annealing, the
Dynabeads-bound template DNA was washed twice with 100 /tl
of the pre-warmed Sequenase reaction buffer, using the MPC
in the water bath to separate the Dynabeads between washes.
Finally, the Dynabeads were resuspended in 10 /tl of 1X
Sequenase reaction buffer and sequenced using the Sequenase
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Figure 1. Sequencing gel showing five sets of sequencingreactions,each containing
identical template DNA and sequencing primer combinations, but differing in
their treatment after sequencing primer annealing. Sequencing template was
prepared as described below. In all cases sequencing primer was annealed to
Dynabead bound template DNA as described in the text. Following this, sequencing
reactions were carried out on sample 1 with no further processing. Samples 2
to 5 were washed as described in the text with the following variations: Samples
2 and 3 were washed once and twice respectively at 45°C and samples 4 and
5 washed once and twice respectivly at 55 °C. Following washing, samples were
resuspended in 10 /d of 1 X Sequenase reaction buffer and sequencing reactions
earned out as with sample 1 using the Sequenase method (4). Samples were run
on a 6% acrylamide sequencing gel (National Diagnostics, Hull, UK) in the order
A, G, C and then T. Sequencing template was part of the mitochondrial D-loop
region between positions 15,749 and 418 of the published sequence of Anderson
et al. (5), amplified from human DNA using the PCR primers HVMlb
(5'-Biotin-CTAACCTGAATCGGAGGACAAC-3f) and HVM4 (5'-GCATACCGCCAAAAGATAAAA-3'). Amplifications were carried out in 100 pi
volumes. PCR products were precipitated by the addition of 1/3 volume 8 M
ammonium acetate and 1 volume isopropanol. Following incubation at room
temperature for 10 minutes and centrifugation at 12,000 g, the samples were
washed once with 70% ethanol, re-dissolved in 15 pi TE (10 mM Tris-HCl
pH 7.5, 1 mM EDTA) and bound to 8 p\ of Dynabeads as described by Hultman
etal. (3). An internal H-strand primer, MT1 (5'-TGGTTAGGCTGGTGTTA-3'),
was used as the sequencing primer.