4016-4017 Nucleic Acids Research, 1994, Vol. 22, No. 19 © 1994 Oxford University Press Ligation of multiple DNA fragments through uracil-DNA glycosylase generated ligation sites Hsiao-Sheng Liu*, Hong-Chang Tzeng, Yuh-Jin Liang and Cheng-Chen Chen 1 Department of Microbiology and Immunology, National Cheng Kung University, Tainan and department 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 chain reaction (PCR) products, is normally mediated by complementary restriction endonuclease sites. Construction of full-length cDNA of a large gene is frequently hampered by the limitation in available ligation sites (1). We developed a simple ligation strategy consisting of PCR with uracil containing primers, bacterial uracil-DNA glycosylase (UDG) digestion, and T4 DNA ligase ligation reaction. It starts with the design of the desired ligation site by replacing the specific thymine-containing nucleotide (T) with the uracil-containing nucleotide (U) in primers by 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 region of the two primers (e.g. _TCGCGA~1- P^-R was then conducted to amplify the DNA fragments with the specific uracil-containing ligation ends. The uracil bases in the PCR amplified products were hydrolyzed by the UDG, and the apurinic or apyrimidinic backbone 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 the fragments at the specific ligation site. To demonstrate the specific ligation, a ligation site which divides the SV40 1.1 kb promoter DNA into 340 and 780 bp fragments was designed (Figure 1). After PCR amplification and UDG treatment at 37 °C for 15 min (4), the two fragments were further treated at 100°C for 15, 30 and 60 min to break the apurinic or apyrimidinic backbone (Figure 2A and 2B, lanes 2,3 and 4). The fragments were then ligated with T4 DNA ligase (BRL, USA) at 25°C for 5 hours (5), and a 1.1 kb ligation product was detected (Figure 3A). To confirm the specificity and accuracy of the ligation, the SV2U and SV3U primers were designed not only to destroy the original PvuU site but also to create a new Haell site (Figure 1). Figure 3B, lane 2 shows the creation of a HaeU site (560, 287, and 253 bps). Figure 3B, lane 7 demonstrates the lost of the PVMII site (1100 bps). To further a + UDG ? 15 30 60 60 12 3 4 5 B 780 bp +UDG UCGCGA TCGCGA 2. Primers SV1 : 5'-> A AGC TTG AGA AAT GGC ATT A <-3' 20 mer SV2U : 5'-> AGC GCU CAC AAT TCC_ TGG TTC TTT CCG <-3' 27 mer SV3U : 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 relative positions of primers. residue - residue • !tL Figure 2. Characterization of UDG cleavage conditions using 340 and 780 bp SV40 DNA fragments. A and B represent 340 and 780 bp fragments, respectively which are PCR amplified and end-labeled with 32 PDATP (5). lane 1: without UDG treatment; lanes 2—4: UDG treatment for 15, 30 and 60 min, individually at 37°C, followed by 100°C boiling for 15 min; lane 5: UDG treatment for 60 min, but without boiling. Residue indicates the removal of cleaved nucleotides. *To whom correspondence should be addressed Downloaded from https://academic.oup.com/nar/article-abstract/22/19/4016/2400214 by guest on 12 February 2018