4016-4017 Nucleic Acids Research, 1994, Vol. 22, No. 19 © 1994 Oxford University Press

Ligation of multiple DNA fragments through uracil-DNAglycosylase generated ligation sites

Hsiao-Sheng Liu*, Hong-Chang Tzeng, Yuh-Jin Liang and Cheng-Chen Chen1

Department of Microbiology and Immunology, National Cheng Kung University, Tainan anddepartment of Parasitology, National Yang-Ming University, Taipei, Taiwan, Republic of China

Received June 9, 1994; Revised and Accepted August 15, 1994

Ligation of DNA fragments, especially the polymerase chainreaction (PCR) products, is normally mediated by complementaryrestriction endonuclease sites. Construction of full-length cDNAof a large gene is frequently hampered by the limitation inavailable ligation sites (1). We developed a simple ligationstrategy consisting of PCR with uracil containing primers,bacterial uracil-DNA glycosylase (UDG) digestion, and T4 DNAligase ligation reaction. It starts with the design of the desiredligation site by replacing the specific thymine-containingnucleotide (T) with the uracil-containing nucleotide (U) in primersby the addition of uracil-phosphoramidite (Cruachem, Scotland)in the DNA synthesizer (PCR-MATE, 391, ABI, California).The replaced T ' is the last nucleotide in the overlapping regionof the two primers (e.g. _TCGCGA~1- P^-R was then conductedto amplify the DNA fragments with the specific uracil-containingligation ends. The uracil bases in the PCR amplified productswere hydrolyzed by the UDG, and the apurinic or apyrimidinicbackbone was then broken by heat to expose the ligation sites

1. SV40 1.1 kb promoter DNA

Hindlll

J

(2,3). Finally, the ligation reaction was conducted to link thefragments at the specific ligation site.

To demonstrate the specific ligation, a ligation site whichdivides the SV40 1.1 kb promoter DNA into 340 and 780 bpfragments was designed (Figure 1). After PCR amplification andUDG treatment at 37 °C for 15 min (4), the two fragments werefurther treated at 100°C for 15, 30 and 60 min to break theapurinic or apyrimidinic backbone (Figure 2A and 2B, lanes 2,3and 4). The fragments were then ligated with T4 DNA ligase(BRL, USA) at 25°C for 5 hours (5), and a 1.1 kb ligationproduct was detected (Figure 3A). To confirm the specificity andaccuracy of the ligation, the SV2U and SV3U primers weredesigned not only to destroy the original PvuU site but also tocreate a new Haell site (Figure 1). Figure 3B, lane 2 shows thecreation of a HaeU site (560, 287, and 253 bps). Figure 3B, lane7 demonstrates the lost of the PVMII site (1100 bps). To further

a + UDG

? 15 30 60 601 2 3 4 5

B

780 bp

+UDG

UCGCGA TCGCGA

2. Primers

SV1 : 5'-> A AGC TTG AGA AAT GGC ATT A <-3' 20 merSV2U : 5'-> AGC GCU CAC AAT TCC_ TGG TTC TTT CCG <-3' 27 merSV3U : 5'-> AGC GCU CAC AAT TCC. TGT GGA ATG TG <-3' 26 mer

SV4 : 5'-> AAG CTT TTT GCA AAA GCC <-3' 18 mer

Figure 1. The schematic map of the 1.1 kb SV40 promoter DNA and relativepositions of primers.

residue - residue • !tLFigure 2. Characterization of UDG cleavage conditions using 340 and 780 bpSV40 DNA fragments. A and B represent 340 and 780 bp fragments, respectivelywhich are PCR amplified and end-labeled with 32PDATP (5). lane 1: withoutUDG treatment; lanes 2—4: UDG treatment for 15, 30 and 60 min, individuallyat 37°C, followed by 100°C boiling for 15 min; lane 5: UDG treatment for 60min, but without boiling. Residue indicates the removal of cleaved nucleotides.

*To whom correspondence should be addressed

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Nucleic Acids Research, 1994, Vol. 22, No. 19 4017

1 2 3 4 1 2 3 4

1100 bp780 bp

340 bp

287,

B

4100 bp

1500bp1489 bp1270 bp

Figure 3. Ligation of the 340 bp and 780 bp DNA fragments and identificationof the 1.1 kb ligation product by PvuU and HaeU restriction endonuclease digestion.A: Lane 1 is the 1100 bp DNA control; lanes 3 and 4 are 1 kb ladder and 123bp marker DNA, respectively; lane 2 shows the ligation of 340 and 780 bp DNAfragments. * indicates the 1100 bp ligation product, and the band above it is theself-ligation of 780 bp DNA fragment. B: Lane 1 and lane 6 are 123 and 1 kbladder markers, respectively; Lane 2: 1.1 kb ligation product after HaeU digestionligation created a new HaeU digestion site, therefore the 560, 287 and 253 bpfragments were detected; Lane 3: Control 1.1 kb fragment after HaeU digestion(847 and 253 bp fragments); lanes 4 and 5 are the undigested 1.1 kb ligationand control products, respectively; lane 7: 1.1 kb ligation product after PvuUdigestion (1100 bp fragment). Ligation destroyed the PvuU digestion site; lane8: Control 1.1 kb DNA fragment after PvuU digestion (780 and 340 bp fragments).

Figure 4. Ligation of dengue-2 virus RTPCR DNA fragments 3, 4 and 5. A:Lane 1 is the RTPCR DNA fragment 3, 1489 bp; lane 2 is the fragment 4, 1270bp and lane 3 is the fragment 5, 1500 bp; lane 4 is the 1 kb ladder marker. B:Lane 1 is the ligation product of RTPCR DNA fragment 3, 4 and 5, and lane2 is the 1 kb ladder marker. * indicates the 4100 bp ligation product.

ACKNOWLEDGEMENTS

This work was supported by grants from National ScienceCouncil, Taiwan, R.O.C. (NSC82-0412-BO06-053 andNSC82-0412-BO06-076-M07).

test the ligation of multiple DNA fragments, three dengue-2 viruscDNA fragments (1270, 1489, and 1500 bps) amplified byreverse transcription PCR (RTPCR) from dengue-2 virus RNA(Figure 4A) were ligated using the same method as describedabove. Figure 4B demonstrates the successful ligation of threefragments together as a 4.1 kb fragment. The ligation sites andthe order of the fragments were checked by DNA sequencing(data not shown).

The unique features of UDG mediated ligation are as follows:1) The sites for the DNA fragment ligation can be anywhere inthe DNA or RNA template and are not restricted by the restrictionenzyme sites. 2) The ligation site, unlike restriction enzyme site,need not be palindromic, so self-ligation can be prevented andthe ligation efficiency can be enhanced. 3) The ligation sitecreated by UDG cleavage, has a protruding 3' end. 4) The ligationand cloning of multiple DNA fragments can be conducted allat once after UDG treatment. This method is extremely usefulfor general cloning and multiple cDNA fragment ligation, e.g.dengue virus or HCV etc.

REFERENCES

1. Lai.C.H., Zhao.B., Hori.H. and Bray,M. (1991) Proc. Nail. Acad. Sd. USA88, 5139-5143.

2. Krokan,H. and Wittwer.C.U. (1993) Nucleic Acid Res. 9, 2599-2613.3. Neddermann.P. and Jiricny.J. (1994) Proc. Natl. Acad. Sci. USA 91,

1642-1646.4. Varshney,U. and Sande.J.H. (1991) Biochemistry 30, 4055-4061.5. Maniatis.T., Fritsch,E.F. and Sambrook.J. (1989) Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

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