Enzymes for Molecular Biology |
Recombination Proteins
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Applications
- Targeting restriction endonuclease digestion to any restriction enzyme
site in cloned ssDNA via DNA oligo splints.1
- Enhancement of the specificity and yield of PCR.2
- Improving the DNA sequencing results through regions with strong secondary
structure.3
- Site-directed mutagenesis when used in conjunction with RecA protein.4
- Improving the processivity of DNA polymerases.
- DNA replication and recombination studies.
- Transcription of ssDNA templates by MiniV™ RNA Polymerase.
E. coli Single-Strand DNA Binding Protein (SSB) binds ssDNA with
high specificity.5,6 In vivo, the protein is involved
in DNA replication, recombination, and repair. In vitro, SSB
enhances several molecular biology applications by destabilizing DNA
secondary structure and increasing the processivity of polymerases.
E. coli SSB is also required for in vitro transcription
of ssDNA templates by MiniV™ RNA Polymerase, a transcriptionally
active 1,106-amino acid domain of the N4 virion RNA polymerase.7 MiniV
RNA Polymerase lacks RNA strand-displacement or unwinding activity on
RNA:DNA hybrids and instead uses E. coli SSB Protein to displace
the RNA transcript from the DNA template strand for efficient in vitro transcription.8
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5),
0.1 mM EDTA, 0.1 M NaCl, 1 mM DTT, and 0.1% Triton® X-100.
Quality Control: Single-Stranded DNA Binding Protein is tested
to be free of detectable endonuclease, DNase, and RNase activities. The
single-strand binding activity of the protein is tested and confirmed
in a gel retardation assay using single-stranded M13mp18 DNA (Fig. 1). |
 |
| Figure 1. SSB efficiently binds ssDNA. Increasing
dilutions of SSB were incubated in 50-µl reactions containing 1 µg
of single-stranded M13mp18 DNA for 30 minutes at 37°C. A 10-µl
aliquot of each reaction was loaded onto an agarose gel and binding of
each dilution of SSB to the DNA was assessed by observing the decreased
mobility of the DNA in the gel. Lane 1, M13mp18 DNA without added SSB;
lane 2, 0.1 µg/µl SSB; lane 3, 0.05 µg/µl
SSB; lane 4, 0.025 µg/µl SSB. |
References
- Milavetz, B. (1989) Nucleic Acids Res. 17, 3322.
- Schwarz, K. et al. (1990) Nucleic Acids Res. 18, 1079.
- Kowalczykowski, S.C. et al. (1981) The Enzymes, 3rd ed.,
Academic Press, New York, NY, 14, 373.
- Shortle, D. (1980) Proc. Natl. Acad. Sci. USA 69, 3537.
- Krauss, G. et al. (1981) Biochemistry 20, 5346.
- Weiner, J.H. et al. (1975) J. Biol. Chem. 250, 1972.
- Kazmierczak, K. M. et al. (2002) EMBO J. 21, 5815.
- Davydova, E. K. and Rothman-Denes, L. B. (2003) Proc. Natl. Acad. Sci.
USA 100, 9250.
MasterAmp™ DNA Polymerases
MiniV™ In Vitro Transcription Kit
RecA Protein, E. coli
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