Description:

Single-Strand Binding Protein (SSB) binds to single-stranded DNA with high affinity and also binds to RNA and double-stranded DNA with lower affinity (1). In vivo, it stabilizes transiently formed ssDNA and plays an important role in DNA replication, recombination and repair (2). In vitro, SSB proteins have been used to destabilize secondary structures in DNA and to increase the processivity of DNA polymerases in several molecular biology applications: SSBs improve the yield and efficiency of reverse transcription reactions during RT-PCR as well as increase the yield of PCR products (3-9).

ET SSB (Extreme thermostable SSB) is a single-stranded DNA binding protein isolated from a hyperthermophilic microorganism, and it is a flagship enhancer of the Primer Navigator product series. It remains fully active after incubation at 95oC for 60 min. Due to the extreme thermostability, ET SSB can be used in applications that require extremely high temperature conditions, such as nucleic acid amplification and sequencing.

Source:

Applications:

Improve the yield of multiplex PCR (Fig.1) and multiplex HDA (Fig.2)
Improve the processivity of DNA polymerase (10)
Stabilization and marking of ssDNA structure (11)
Increase the yield and specificity of PCR reactions (5-9)
Increase the yield and processivity of RT during RT-PCR (3,4)
Improve DNA sequencing through regions with strong secondary structure (8)
Enhance the RecA activity for ssDNA binding and strand trasfer (12,13)

Storage Conditions:

Storage Buffer:
20 mM Tris-HCl
200 mM NaCl
1 mM EDTA
0.5 mM DTT
50% Glycerol
pH7.5 @ RT

Storage Temperature:
-20oC

Quality Control:

Quality Assurance Statement:
ET SSB is purified free of contaminating endonucleases and exonucleases. Each lot is tested for single-strand, DNA-dependent ATPase activity and is visually determined to be > 95% pure on an SDS-polyacrylamide gel.

Exonuclease Activity:
Incubation of 20 μg ET SSB for 4 hours at 37°C in 50 μl reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg of a mixture of single and double-stranded [³H] E. coli DNA (200,000 cpm/μg) released < 0.05% of the total radioactivity.

Endonuclease Assay:
Incubation of 10 μg ET SSB for 4 hours at 37°C in 50 μl reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg ΦX174 RF I DNA gave < 5% conversion to RF II.

Nuclease Activity:
Incubation of 20 μg ET SSB for 16 hours at 37°C in 50 μl of reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg λ DNA yielded a clear and sharp band on an agarose gel.

References:

1. Coleman, J. E. & Oakley, J. L. (1980) CRC Crit Rev Biochem 7, 247-89.
2. Chase, J. W. & Williams, K. R. (1986) Annu Rev Biochem 55, 103-36.
3. Baugh, L. R., Hill, A. A., Brown, E. L. & Hunter, C. P. (2001) Nucleic Acids Res 29, E29.
4. Villalva, C., Touriol, C., Seurat, P., Trempat, P., Delsol, G. & Brousset, P. (2001) Biotechniques 31, 81-3, 86.
5. Schwarz, K., Hansen-Hagge, T. & Bartram, C. (1990) Nucleic Acids Res 18, 1079.
6. Chou, Q. (1992) Nucleic Acids Res 20, 4371.
7. Oshima, R. G. (1992) Biotechniques 13, 188.
8. Rapley, R. (1994) Mol Biotechnol 2, 295-8.
9. Olszewski, M., Rebala, K., Szczerkowska, Z. & Kur, J. (2005) Mol Cell Probes 19, 203-5.
10. Myers, T. W. & Romano, L. J. (1988) J Biol Chem 263, 17006-15.
11. Delius, H., Mantell, N. J. & Alberts, B. (1972) J Mol Biol 67, 341-50.
12. Reddy, M. S., Vaze, M. B., Madhusudan, K. & Muniyappa, K. (2000) Biochemistry 39, 14250-62.
13. West, S. C., Cassuto, E. & Howard-Flanders, P. (1982) Mol Gen Genet 186, 333-8.ET SSBH0221S