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DNA Cloning and Engineering by Uracil Excision
Publication Name:
Current Protocols in Molecular Biology
Unit Number:
Unit 3.21
DOI:
10.1002/0471142727.mb0321s86
Online Posting Date:
April, 2009 Abstract
This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single-stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment. Two basic protocols on the methods of uracil excision-based engineering are presented, and special attention is given to primer design. The use of a commercially available cloning vector and the preparation of custom vectors are also presented. Curr. Protoc. Mol. Biol. 86:3.21.1-3.21.16. © 2009 by John Wiley & Sons, Inc.
Keywords: uracil excision; UDG; USER enzyme; DNA mutagenesis; directional cloning; DNA assembly; nicking endonuclease
Table of Contents
- Introduction
- Basic Protocol 1: General Methods for DNA Cloning and Engineering by Uracil Excision
- Basic Protocol 2: Construction and Linearization of Custom-Made Vectors Compatible with DNA Cloning by Uracil Excision
- Alternate Protocol 1: Site-Specific Mutagenesis
- Alternate Protocol 2: Sequence Insertions
- Alternate Protocol 3: Sequence Deletions
- Alternate Protocol 4: Nucleotide Sequence Assembly, Domain Substitution, and Multiple Sequence Manipulations
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol 1: General Methods for DNA Cloning and Engineering by Uracil Excision
Materials
- Template DNA
- Oligonucleotide primers, each containing a single dU residue (see discussion on primer design, above; see units
- Pfu Turbo C
x Hotstart DNA Polymerase (Stratagene) and 10× Pfu Cx Reaction Buffer (Stratagene) - 20 U/µl restriction endonuclease DpnI
- 20 ng/µl linearized vector pNEB206A (Fig.
- 1 U/µl USER Enzyme (New England Biolabs)
- Chemically competent E. coli cells (available from various molecular biology suppliers; also see unit
- 2 U/µl restriction endonuclease BbvCI and 10× buffer for BbvCI
- Additional reagents and equipment for DNA amplification by PCR (unit
Basic Protocol 2: Construction and Linearization of Custom-Made Vectors Compatible with DNA Cloning by Uracil Excision
Materials
- 0.1 pmol appropriate phosphorylated, blunt-ended DNA vector
- 2.5 pmol/µl Universal USER Cassette (New England Biolabs)
- T4 DNA Ligase (2000 U/µl; New England Biolabs) and 10× T4 DNA ligase buffer
- Chemically competent E. coli cells (available from various molecular biology suppliers; also see unit
- 10× XbaI buffer
- Restriction endonuclease XbaI
- Nt.BbvCI nicking endonuclease (New England Biolabs)
- TE buffer, pH 8.0 (appendix
- Additional reagents and equipment for transformation of DNA (unit
Looking for materials? Suppliers Guide
Figures
-
Figure 3.21.1Schematic description of DNA engineering method via uracil excision. PCR primers used in the same amplification reaction match in shading. “U” stands for deoxyuracil. Primers P1 and P2 precisely overlap each other in the sequence upstream of a U. The specific sequences GGAGACAU in the Left primer and GGGAAAGU in the Right primer are designed to be compatible with the pNEB206A vector (see Fig. -
Figure 3.21.2General guidelines for designing PCR primers to be used with uracil excision–based DNA engineering method. The junction sequence, ANNNNNNT, within the template is boxed. Primers P1 and P2 precisely overlap each other by the junction sequence except for the replacement of a 3¢ deoxythymine with a dU in the primer. Templates and their respective primer pair match in shading. “N” stands for any nucleotide; “U” stands for deoxyuridine. -
Figure 3.21.3Schematic representation of the pNEB206A cloning vector. (A) pNEB206A vector was constructed by ligating a synthetic double-stranded cassette into the PacI and PmeI sites of pNEB193 (New England Biolabs). Within the cassette, XbaI and Nt.BbvCI recognition sequences are underlined; cleavage sites are shown by arrows. (B) For cloning by uracil excision, the pNEB206A is double-digested with XbaI and Nt.BbvCI to produce linearized vector flanked by 3¢ single stranded extensions on both ends. -
Figure 3.21.4PCR primer design for site-specific mutagenesis via uracil excision. In the primers, the desired sequence change is shown as “X”. (A) Sequence changes can be integrated into both overlapping primers P1 and P2 within the junction sequence. (B) Sequence changes can be integrated into one overlapping primer, e.g., P1 downstream from the junction sequence. -
Figure 3.21.5PCR primer design for sequence insertions via uracil excision. (A) Shown is the location of the intended insertion sequence (colored dark gray) within target DNA. Pseudo-template sequence files carrying insertion sequence are created to find the best-suited junction sequences and to design primers P1 and P2 for short insertions (B), and for long insertions (C). -
Figure 3.21.6PCR primer design for sequence deletions via uracil excision. (A) Shown in the darkest shading is the nucleotide sequence intended to be deleted from target DNA. (B) A pseudo-template DNA sequence file lacking the deletion sequence is created to find the best-suited junction sequence and to design primers P1 and P2. (C) Primers P1 and P2 prime distant locations on the template, but overlap each other in junction sequence, ANNNNNNU. -
Figure 3.21.7PCR primer design for multiple fragment assembly via uracil excision. (A) Shown in white, light gray and dark gray are target DNAs 1, 2, and 3, respectively, that will be assembled. (B) A pseudo-template DNA sequence file containing the assembled sequence is created to find the best-suited junctions and to design the primer pairs P1/P2 and P3/P4. (C) Primers P2 and P1 prime template 1 and template 2, and overlap each other in the junction sequence, ANNNNNNU. Primers P4 and P3 prime template 2 and template 3, and overlap each other in junction sequence, AnnnnnnU. Three separate PCR reactions are performed using templates 1, 2, and 3 with primer pairs Left/P2, P1/P4 and P3/Right, respectively.
Literature Cited
| Literature Cited | |
| Bitinaite, J., Rubino, M., Hingorani-Vaarma, K., Schildkraut, I., Vaisvila, R., and Vaiskunaite, R. 2007. USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35:1992-2002. | |
| Braman, J., Papworth, C., and Greener, A. 1996. Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol. Biol. 57:31-44. | |
| Booth, P.M., Buchman, G.W., and Rashtchian, A. 1994. Assembly and cloning of coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase. Gene 146:303-308. | |
| Fogg, M.J., Pearl, L.H., and Connolly, BA. 2002. Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat. Struct. Biol. 9:922-927. | |
| Greagg, M.A., Fogg, M.J., Panayotou, G., Evans, S.J., Connolly, B.A., and Pearl, L.H. 1999. A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. Proc. Natl Acad. Sci. U.S.A. 96:9045-9050. | |
| Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G., and Galas, D.J. 1989. A simple method for site-specific mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 17:6545-6551. | |
| Ho, S.F., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. 1989. Site-directed mutagenesis by overlap extension using polymerase chain reaction. Gene 77:51-59. | |
| Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77:61-68. | |
| Lasken, R.S., Schuster, D.M., and Rashtchian, A. 1996. Archaebacterial DNA polymerases tightly bind uracil-containing DNA. J. Biol. Chem. 271:17692-17696. | |
| Lundberg, K.S., Shoemaker, D.D., Adams, M.W.W., Short, J.M., Sorge, J.A., and Mathur, E.J. 1991. High-fidelity amplification using thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1-6. | |
| Nisson, P.E., Rashtchian, A., and Watkins, P.C. 1991. Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Methods Appl. 1:120-123. | |
| Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H., Jensen, J.K., and Halkier, B.A. 2006. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34:e122. | |
| Rashtchian, A., Thornton, C.G., and Heidecker, G. 1992. A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase. PCR Methods Appl. 2:124-130. | |
| Weiner, M.P., Costa, G.L., Schoetlin, W., Cline, J., Mathur, E., and Bauer, J.C. 1994. Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151:119-123. | |
| Key Reference | |
| Bitinaite et al., 2007. See | |
| Provides specific examples of DNA engineering by uracil excision. | |
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