Abstract
Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gayle R. B., 3rd, Auger E. A., Gough G. R., Gilham P. T., Bennett G. N. Formation of MboII vectors and cassettes using asymmetric MboII linkers. Gene. 1987;54(2-3):221–228. doi: 10.1016/0378-1119(87)90490-2. [DOI] [PubMed] [Google Scholar]
- Kegler-Ebo D. M., Docktor C. M., DiMaio D. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes. Nucleic Acids Res. 1994 May 11;22(9):1593–1599. doi: 10.1093/nar/22.9.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim S. C., Szybalski W. Amplification of cloned DNA as tandem multimers using BspMI-generated asymmetric cohesive ends. Gene. 1988 Nov 15;71(1):1–8. doi: 10.1016/0378-1119(88)90071-6. [DOI] [PubMed] [Google Scholar]
- Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
- Montgomery J., Goldman S., Deikman J., Margossian L., Fischer R. L. Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5939–5943. doi: 10.1073/pnas.90.13.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Odell J. T., Nagy F., Chua N. H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. 1985 Feb 28-Mar 6Nature. 313(6005):810–812. doi: 10.1038/313810a0. [DOI] [PubMed] [Google Scholar]
- Ow D. W., DE Wet J. R., Helinski D. R., Howell S. H., Wood K. V., Deluca M. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science. 1986 Nov 14;234(4778):856–859. doi: 10.1126/science.234.4778.856. [DOI] [PubMed] [Google Scholar]
- Ståhl S., Sjölander A., Hansson M., Nygren P. A., Uhlén M. A general strategy for polymerization, assembly and expression of epitope-carrying peptides applied to the Plasmodium falciparum antigen Pf155/RESA. Gene. 1990 May 14;89(2):187–193. doi: 10.1016/0378-1119(90)90005-c. [DOI] [PubMed] [Google Scholar]
- Szybalski W., Kim S. C., Hasan N., Podhajska A. J. Class-IIS restriction enzymes--a review. Gene. 1991 Apr;100:13–26. doi: 10.1016/0378-1119(91)90345-c. [DOI] [PubMed] [Google Scholar]