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The Department of Oncology, The Children's Hospital, Ladywood Middleway, ... Group, University Department of Clinical Chemistry, Wolfson Research.
.=) 1991 Oxford University Press

Nucleic Acids Research, Vol. 19, No. 24 6959

Sequencing self-ligated PCR products using 3' over-hangs generated by specific cleavage of dUTP by uracil-DNA glycosylase P.J.R.Day and M.R.Walkerl The Department of Oncology, The Children's Hospital, Ladywood Middleway, Birmingham B16 8ET, 'Molecular Biology Research Group, University Department of Clinical Chemistry, Wolfson Research Laboratories, The Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, UK Submitted July 9, 1991 Several methods have been suggested for improving direct sequencing of PCR products (1, 2, 3) reflecting the difficulty of sequencing most amplified DNA. We have noted doublestranded plasmids are more amenable to direct sequencing compared to PCR generated products. Because inhibition of sequencing primer annealing is believed to result from the rapid rate of PCR strands reannealing (4, 5), we have circularised PCR products to discourage premature chain terminations (6). PCR products were self-ligated using homopolymer tails generated by the inclusion of 5' polyadenosine and polythymidine sequences followed by deoxyuracil (dUTP) to the forward and reverse primer sequences respectively. We were able to specifically excise dUTP from the PCR primers by using uracil-DNA glycosylase to leave complementary sequences to the homopolymer sequences single-stranded. The homopolymer sequences were chosen to be complementary, and were used to facilitate efficient ligation and subsequent sequencing of 120 base pair and 710 base pair PCR products. Control reactions omitting ligase did not produce a usable sequencing template. Therefore, because concatamerisation was not observed, the conformation of PCR product formed in the presence of DNA ligase must reduce the rate of PCR product reannealing, thus assisting annealing of the sequencing primer and enabling direct sequencing from either PCR strand. The ligation strategy is outlined in Figure 1. 1 jtM of each primer (possessing an additional 5' sequence of 9 bases of either dATP or dTTP plus a dUTP base) were used in standard 100 Al PCR reaction mixes, and subjected to 30 amplification cycles comprising 80°C for 30 sec, 40°C for 30 sec and 72°C for 60 sec. Following amplification, 1 unit of uracil-DNA glycosylase (Perkin-Elmer-Cetus) is added, incubated at 18°C for 15 min, heat denatured and quickly loaded onto an agarose gel for band excision, elution and ethanol precipitation. Ligation is performed overnight at 4°C, phenol and ethanol extracted, and the purified circular fragments are resuspended in 20 I1I of TIOE1, pH 8.0. Sequencing is performed using 3 11 of the fragment, 1 Iul of reaction buffer (6 x reaction buffer: 240 mM Tris-HCl, pH 7.5, 150 mM MgCl2, 300 mM NaCl and 6% DMSO) and 2 A1 of primer at 10 ,uM (can be an amplimer or a mid-sequence oligonucleotide), boiled for 3 minutes and quenched in liquid of each of 100 mM DTT and 35S dATP (SJ 1304, N2. 1 Amersham International, plc, Bucks, UK) are added to the thawing mixture, followed by 2 units of Sequenase (United States

Biochemical, Cleveland, OH), mixed and 2 Al samples placed in each termination mixture (Sequenase Kit, USB, made 10% for DMSO) and incubated for 5 minutes each at room temperature and 37°C. 2 Al of chase mixture (0.25 mM dNTPs, 50 mM NaCl and 10% DMSO) are added into each tube and incubated for 15 min, before addition of 4 Al of formamide stop buffer. The introduction of an A-A mismatch does not impose a problem for ligation, presumably due to the clamping produced by the polymonobasic sequence. Also, the poor fidelity with which polymerases read dUTP may actually increase the likelihood of correctly matched sequences, although this effect has not been observed. By introducing one or more over-hanging bases into PCR products (as depicted by primer sequence 5' to the uracil moiety), all amplified products will have the appropriate over-hang, rather than relying on the chance probability of nontemplate directed 3' incorporation of adenosine by Taq polymerase (7).

ACKNOWLEDGEMENTS We would like to thank the British Technology Group for funding this work.

REFERENCES 1. Gyllensten,U.B. (1989) in Erlich,H.A. (ed.) PCR Technology. Stockton Press, pp. 45-60. 2. Sarker,G. and Sommer,S.S. (1988) Nucleic Acids Res. 16, 5197. 3. Gyllensten,U.B. and Erlich,H.A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652-7656. 4. Winship,P.R. (1989) Nucleic Acids Res. 17, 1266. 5. Casanova,J.-L. et al. (1990) Nucleic Acids Res. 18, 4028. 6. Lee,J.-S. (1991) DNA and Cell Biol. 10, 67-73. 7. Holton,T.A. and Graham,M.W. (1991) Nucleic Acids Res. 19, 1156. Forward primer E..l

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See text for ligation strategy.