Polymerases and Amplification Technologies
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  • DNA Manipulation

    Bsu DNA Polymerase I, Large Fragment (NEB #M0330)

    Bsu DNA Polymerase I, Large Fragment retains the 5´→ 3´ polymerase activity of the Bacillus subtilis DNA polymerase I (1), but lacks the 5´→ 3´ exonuclease domain. This large fragment naturally lacks 3´→ 5´ exonuclease activity.

    DNA Polymerase I (E. coli) (NEB #M0209)

    DNA Polymerase I (E. coli) is a DNA-dependent DNA polymerase with inherent 3´→ 5´ and 5´→ 3´ exonuclease activities (2). The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation.

    DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210)

    DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I, which retains polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity (3). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

    Klenow Fragment (3´→ 5´ exo- ) (NEB #M0212)

    Klenow Fragment (3´→ 5´ exo- ) is an N-terminal truncation of DNA Polymerase I, which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has mutations (D355A, E357A), which abolish the 3´→ 5´ exonuclease activity (4).

    phi29 DNA Polymerase (NEB #M0269)

    phi29 DNA Polymerase is the replicative polymerase from the Bacillus subtilis phage, phi29 (Φ29) (5). This polymerase has exceptional strand displacement and processive synthesis properties (6). The polymerase has an inherent 3´→5´ proofreading exonuclease activity (7).

    T4 DNA Polymerase (NEB #M0203)

    T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. This enzyme has a 3´→ 5´ exonuclease activity, which is much more active than that found in DNA Polymerase I. Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5´→ 3´ exonuclease function.

    T7 DNA Polymerase (unmodified) (NEB #M0274)

    T7 DNA Polymerase (unmodified) catalyzes the replication of T7 phage DNA during infection. The protein dimer has two catalytic activities: DNA polymerase activity and strong 3´→ 5´ exonuclease activity (8,9,10). The high fidelity and rapid extension rate of the enzyme make it particularly useful in copying long stretches of DNA template.

    Terminal Transferase (NEB #M0315)

    Terminal Transferase (TdT) is a template-independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. Protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as a substrate for TdT. The 58.3 kDa enzyme does not have 5' or 3' exonuclease activity. The addition of Co2+ in the reaction makes tailing more efficient.

    References

    1. Okazaki, T. et al. (1964) J. Biol. Chem., 239, 259-268. PMID: 14114852
    2. Lehman, I.R.. (1981) P.D. Boyer (Eds.), The Enzymes, 14A, pp.16-38. San Diego: Academic Press.
    3. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974) Eur. J. Biochem., 45, 623-7. PMID: 4605373
    4. Derbyshire, V. et al. (1988) Science, 240, 199-201. PMID: 1309614
    5. Blanco, L. and Salas, M. (1984) Proc. Natl. Acad. Sci. USA, 81, 5325-9. PMID: 6433348
    6. Blanco, L. et al. (1989) J. Biol. Chem., 264, 8935-40. PMID: 2498321
    7. Garmendia, C., et al. (1992) J. Biol. Chem., 267, 2594-9. PMID: 1733957
    8. Hori, K. et al. (1979) J. Biol. Chem., 258, 11598-11604. PMID: 227873
    9. Engler, M.J. et al. (1983) J. Biol. Chem., 258, 11165-73. PMID: 6411726
    10. Nordstrom, B. et al. (1981) J. Biol. Chem., 256, 3112-7. PMID: 7009606

    Applications

    • Random primer labeling 
    • Second strand cDNA synthesis 
    • Strand displacement DNA synthesis (1) 
    • Nick translation of DNA to obtain probes with a high specific activity (2)
    • DNA sequencing by the Sanger dideoxy method (3) • Fill-in of 5´ overhangs to form blunt ends (4) • Second strand synthesis in mutagenesis protocols (5). • Removal of 3´ overhangs to form blunt ends (4)
    • Single strand deletion subcloning (6).
    • Addition of homopolymer tails to the 3' ends of DNA
    • Labeling the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP)
    • TUNEL assay (in situ localization of apoptosis)
    • TdT dependent PCR