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© Oxford University Press 1990
Nucleic Acids Research, Vol 18, No. 4
Direct sequencing of double-stranded DNA PCR products
via removing the complementary strand with singlestranded DNA of an M13 clone
Susannah Gal and Barbara Hohn
Friedrich Miescher-lnstitute, PO. Box 2543, CH-4002 Basel, Switzerland
Submitted December 29, 1989
The polymerase chain reaction (PCR (1)) is used to amplify
specific sequences from complicated mixtures of DNA samples.
It is often preferable to sequence these PCR products directly.
Direct sequencing of double-stranded (ds) DNA products has been
reported (2,3,4). There also exist several methods for making
better quality sequencing templates by generating single-stranded
(ss) DNA (reviewed in 5), but these procedures add steps to the
preparation of the samples prior to the actual sequencing reactions
and/or often reduce the yield of the sequencing template. In our
procedure, we create the ssDNA sequencing template in the same
tube during the normal denaturation step by adding an excess
of competing, complementary ssDNA. We found the process
efficient and simple. The procedure requires the cloning of one
variant of the sequence into an M13-based vector system which
will be used for all subsequent sequencing of the related
fragments. The primers for the PCR and for sequencing are
located outside of the cloned region so that the M13 ssDNA has
no homology to the primer.
PCR products were formed from DNA from transgenic
Brassica napus plants expressing the cauliflower mosaic virus
(CaMV) using virus specific primers. The approxiamtely 1000
bp products were purified by spin-dialysis in the Centricon-30
(Amicon) and quantitated by ethidium bromide staining.
Approximately 0.5 pmole of the dsDNA was put into an
Eppendorf tube, denatured with NaOH for 5 min, neutralized
with sodium acetate, and 20 pmole of primer and various amounts
of complementary M13 ssDNA were added. The samples were
then precipitated with ethanol, dried briefly, and taken up in 10
fil sequencing buffer (Sequenase® sequencing kit, US
Biochemicals). The DNA was denatured and reannealed by
incubating at 68°C for 5 min and then allowed to cool slowly
to room temperature over the next 30 minutes. The sequencing
protocol was then carried out as per the manufacturer's
instructions and the samples were loaded onto a standard
sequencing gel. The figure shows the result when increasing
amounts of complementary ssDNA were added to the sequencing
reactions. One sees a dramatic improvement in the sequencing
ladder when approximately 5-fold molar excess of ssDNA is
added. This ladder did not improve when a 10-fold excess was
added (not shown). This simple procedure presumably works by
removing the competing strand of DNA, which has a much higher
affinity than the sequencing primer for the template strand, and
allows the sequencing primer to prime the sequence reactions.
We are using this method to sequence viruses from various
transgenic plants. This procedure will undoubtedly be useful for
screening other virus isolates and in analyzing allelic and
mutational differences in genes when cloning of fragments is
either unnecessary or undesirable. It might also allow one to
selectively sequence one allele in a mixture by simply adding
complementing ssDNA only to that specific allele.
ACKNOWLEDGEMENT
S.G. is supported by a grant from the National Science
Foundation, USA.
REFERENCES
1
2
3
4
5
Saiki.R.K et al (1988) Science 239, 487-491
Kretz.K A el al (1989) Nucl Acids Res 17, 5864
Wong.C. et al (1987) Nature 330, 384-386
Puchta.H and Sacnger,H L (1989) Arch Virol 106, 335-340
Gyllensten.U B (1989) BioTechniques 7, 700-708
•s 1
• I'
Figure 1. Sequencing reactions using dsDNA PCR products and the Sequenase*
sequencing kit and adding increasing amounts of ssDNA, 0.5 pmole dsDNA alone
(panel 1), with 0 5 pmole M13 ssDNA (panel 2), with 2 5 pmole M13 ssDNA
(panel 3) Reactions loaded in the order A,C,G,T.