tandemly repeated DNA sequences with defined length, orientation and spacing. Shi-Wen Jiang1, Miguel A. Trujillo1 and Norman L. Eberhardt1,2,*. 1Endocrine ...
1996 Oxford University Press
3278–3279 Nucleic Acids Research, 1996, Vol. 24, No. 16
An efficient method for generation and subcloning of tandemly repeated DNA sequences with defined length, orientation and spacing Shi-Wen Jiang1, Miguel A. Trujillo1 and Norman L. Eberhardt1,2,* 1Endocrine
Research Unit, Department of Medicine and 2Department of Biochemistry/Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA Received March 4, 1996; Revised and Accepted July 5, 1996
ABSTRACT Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment. Since concatenation of DNA elements increases the affinity for transcriptional factors (1,2), tandemly repeated DNA sequences are used for a variety of purposes, including functional characterization of transactivating factors (3–5), purification of transcriptional factors by DNA affinity column chromatography (1), Southwestern blot analysis and expression library screening (6). The generation of repeated sequences is based on the direct ligation of synthetic monomers. Since relatively high monomer concentrations are involved in these reactions, the phosphorylation and ligation reactions are inefficient and a majority of the ligation products are short oligomers. The small yields of longer oligomers makes their manipulation difficult and the subcloning efficiency of long oligomers is characteristically low. Moreover, the end products of these reactions are random structures containing tandem and inverted repeats. To avoid these problems, we developed a ligation-PCR protocol to generate long DNA fragments containing numerous tandem repeats. With this methodology the length, orientation and spacing of the repeat units may be precisely manipulated. The protocol utilizes double-stranded ‘core’ monomers containing any desired motif (e.g., enhancer element), and linkers containing desired restriction sites. Both oligonucleotides contain
* To
specific overhangs to control orientation during ligation. Ligation is performed with certain ratios of core monomers to linkers to control the repeat number. Ligation products are size-selected, PCR amplified using single-stranded linkers as primers, and restricted to generate DNA fragments carrying sticky ends suitable for ligation without further modification. We have successfully subcloned six individual enhansons from the chorionic somatomammatropin gene enhancer (7) as tandem repeats. Pairs of complementary oligonucleotides containing 5′-GG-3′ and 5′-CC-3′ overhangs and various enhanson sequences were synthesized (e.g., IR1: 5′-AGGATGTTTTCTAAACGATGG, 5′-ATCGTTTAGAAAACATCCTCC). The 19 bp linkers also contain 5′-GG/CC overhangs and SalI–PstI–BglII sites (5′-CGTCGACTGCAGATCTCGG, 5′-GAGATCTGCAGTCGACGCC). Single-stranded oligonucleotides (2 µg) were phosphorylated separately in 8 µl reactions (37C × 90 min) containing 1× phosphorylation buffer, 1 mM ATP and 8 U polynucleotide kinase (Promega). For annealing, each pair of single-stranded oligonucleotides was combined, heated to 90C and slowly cooled to room temperature. After brief centrifugation to collect water condensed on the sides of the tubes, 2 µl linkers were added to the phosphorylated monomers (monomer:linker ratio = 10:1). Ligation was initiated by adding 2 µl 10× ligation buffer and 1 µl (3 U) T4 DNA ligase (Promega) directly to 18 µl of annealed oligonucleotides. After incubation at room temperature for 4 h, half of the ligation product was resolved on a 2% agarose gel. The majority of the ligation products are dimers and trimers and DNA bands representing larger products are barely visible (Fig. 1A). Gel slices containing products of ∼600 bp were subjected to one freeze–thaw cycle. After centrifugation (14 000 g for 5 min), 2 µl of the supernatant (2% of the total volume) was used as a PCR template. The PCR mixture consisted of 10 µl 10× Taq Extender buffer (Strategene), 2 µl 10 mM dNTPs, 0.2 µg of each linker as primers in a volume of 98 µl. After denaturation (94C for 5 min), 1 µl (5 U) of Taq polymerase (Boeringer Mainheim) and 1 µl (5 U) PCR Extender (Strategene) were added and subjected to 35 cycles according to the following protocol: denaturation at 93C for 1 min, annealing at 56C for 1 min, extension at 72C for 3 min, and terminal extension at 72C for 10 min. PCR products were extracted once with phenol– chloroform, ethanol precipitated and redissolved in 50 µl water.
whom correspondence should be addressed at: 4-407 Alfred, SMH, Mayo Clinic, Rochester, MN 55905, USA
3279 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.116 Nucleic
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Figure 1. DNA samples from different stages resolved on 2% agarose gels. DNA markers (bp) are indicated. (A) Phosphorylated double-stranded IR1:linker::10:1 after 4 h ligation. The region isolated for PCR amplification is indicated (parentheses). (B) Products after PCR amplification are extremely large oligomers. (C) BglII-digested PCR products. The 100–200 bp DNA band (parentheses) was isolated for ligation to pA3LUC.
Extremely large PCR products were generated by PCR (Fig. 1B). Similar results were obtained in identical PCR reactions that lacked primers, indicating that self-priming has occurred (data not shown). PCR products (25 µl) were digested with 40 U BglII at 37C, resulting in a heterodisperse collection of fragments most of which are smaller than the size-selected template (Fig. 1C). This results because these templates contain multiple, randomly inserted linkers that will generate concatemers corresponding to dimers, trimers, tetramers, etc., upon BglII digestion. At this stage the DNA oligomers can be ligated directly to appropriate vectors; however, gel isolation/size-selection of DNA fragments facilitates subcloning of inserts with a desired size. In Figure 1C, 100–200 bp DNA fragments (4–8mers) was eluted by Geneclean (BIO101) and ligated to BglII-digested pA3LUC vector (8). After transformation, 12 independent colonies were screened with BglII (Fig. 2A). DNA from selected colonies was purified by CsCl ultracentrifugation and the orientation and number of tandem repeats was ascertained by sequencing (Fig. 2B and C). During the PCR reaction, synthesis directed by the linkers contributed to amplification of individual repeat units, whereas self-priming resulted in the generation of increasingly higher molecular weight products. We also observed that addition of the Taq Extender significantly increased the yield of long oligomers (data not shown). The size of the oligomers is dependent on the ratio of core monomers to linkers used in the ligation reaction. When a high monomer/linker ratio is used, DNA fragments containing as many as 100 head-to-tail repeats can be easily generated (data not shown). This new method allows the generation of tandemly repeated DNA sequences whose size, orientation and spacing can be controlled. In addition to low copy number repeats, useful for transfection studies, it is also capable of generating high copy number polymers which can be utilized to increase the efficiency of DNA affinity resins and DNA probes.
Figure 2. Escherichia coli HB101 was transformed using the ligation-PCR products (Fig. 1C). (A) Colony screening by BglII digestion, indicating various single and multiple inserts. (B) DNA from five clones (A) were purified by double CsCl ultracentrifugation and sequenced. The exact sequence of clone 5 is shown; tandem repeats (bold, arrow), linkers (normal) and BglII sites (underlined) are indicated. (C) Schematic sequence summary of the number and orientation of the tandem repeats (arrows) and linkers (open box) from designated clones (A).
ACKNOWLEDGEMENTS The authors wish to express their appreciation to Drs Stefan Grebe, Andrew M. Arnold and James Levine for helpful discussions and Dr William Wood for providing the pA3LUC vector. REFERENCES 1 Kadonaga, J.T. and Tjian, R. (1986) Proc. Natl. Acad. Sci. USA 83, 5889–5893. 2 Zhang, W.-W., Farres, J. and Busch, H. (1991) BioTechniqes 11, 728–733. 3 Veldman, G.M., Lupton, S. and Kamen, R. (1985) Mol. Cell. Biol. 5, 649–658. 4 Dana, S.L., Hoener, P.A., Wheeler, D.A., Lawrence, C.B. and McDonnell, D.P. (1994) Mol. Endocrinol. 8, 1193–1207. 5 Schafer, A.J. and Fournier, R.E. (1992) Somat. Cell. Mol. Gen. 18, 571–581. 6 Maniatis, T., Fritsch, E. and Sambrook, J. D. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 12–30. 7 Jiang, S.W. and Eberhardt, N.L. (1994) J. Biol. Chem. 269, 10384–10392. 8 Maxwell, I.H., Harrison, G.S., Wood, W.M. and Maxwell, F. (1989) Biotechniques 7, 276–280.
