Abstract
Genomic libraries represent a powerful resource for genetic studies of bacteria. Large-insert libraries in bacterial artificial chromosome (BAC) vectors are particularly important not only for genome-sequencing projects, but for comparative and functional genomic studies once a complete sequence is known. Cloned DNA can be introduced into bacterial cells in two different ways. One way is to have cells take up naked DNA; this is known as transformation (1). The other way is to package the recombinant DNA inside bacteriophage particles in vitro using a phage such as λ. This process, known as in vitro packaging, allows DNA to be introduced by infection (2). Although transformation allows DNA of any size to be introduced, DNA uptake occurs at very low frequency. On the other hand, in vitro packaging limits the size of the DNA based on the phage head stability. However, in vitro package is the most efficient method for introducing large-insert clones.
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References
Zhu, H. and Dean, R. A. (1999) A novel method for increasing the transformation efficiency of Escherichia coli—application for bacterial artificial chromosome library construction. Nucleic Acid Res. 27, 910–911.
Champness, W. and Snyder, L. (1997) Molecular Genetics of Bacteria. American Society of Microbiology.
Weinstock, G. M., Maurer, R., and Berget, P. B. (1990) Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Limberger, J. R., Silvenski, L. L., Izard, J., and Samsonoff, A. W. (1999) Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J. Bacteriol. 181(12), 3743–3750.
Murray, M. G. and Thompson, W. F. (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res. 8, 4321–4325.
Sambrook, J., Fritsch, E. F, and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Ausubel, F M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G, Smith, J. A., and Straw, K. (1989) Current Protocols in Molecular Biology. John Wiley, New York.
Murray, E. M, Singh, K. V., Ross, R. P., Heath, J. D., Dunny, G. M., and Weinstock, G M. (1993) Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J. Bacteriol. 175(16), 216–5222.
Teng, F, Murray, E. B., and Weinstock, G M. (1998) Conjugational transfer of plasmid DNA from E. coli to Enterococcus: A method to make insertional mutation. Plasmid 39, 182–186.
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© 2004 Humana Press Inc., Totowa, NJ
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Pal, S., Mak, S., Weinstock, G.M. (2004). Preparation of BAC Libraries From Bacterial Genomes by In Vitro Packaging. In: Zhao, S., Stodolsky, M. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology™, vol 255. Humana Press. https://doi.org/10.1385/1-59259-752-1:057
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DOI: https://doi.org/10.1385/1-59259-752-1:057
Publisher Name: Humana Press
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