26 Maunders
- Experimental Procedures
T4 DNA polymerase will function in most restriction endonuclease
buffers. An alternative reaction buffer is 50 mMTris-HC1, pH 8.5, 15 mM
ammonium sulfate, 7 mMMgC12, 0.1 mM EDTA, 10 mM ~-mercapto-
ethanol, 33 ~,/dNTPs, and 200 pg/mL BSA (15). The enzyme requires
Mg 2+ (at least 6 mM) for activity. Its pH optimum is in the range
8.0-9.0, exhibiting 50% activity at pH 7.5 and 9.7. The reaction tem-
perature should be 37°C.
The enzyme is best stored in 10 mM Tris-HC1, pH 8.0, 100 mM
NaC1, 2.5 mM MgC12, 0.5 mM EDTA, 2 mM DTT, and 50% gly-
cerol (15). It is stable if stored undiluted in this buffer at -20°C for
several months.
- Experimental Procedures
- T7 DNA Polymerase
This DNA polymerase is also produced by bacteriophage-infected
E. coli, although it is now obtained from a cloned gene.
5.1. Enzyme Data
It is the only common DNA polymerase that consists of more than
one subunit. The functional enzyme is composed of the T7 gene 5
protein (mol mass 80,000-85,000 Da), which carries all the enzymic
activities, plus the E. coli thioredoxin protein (mol mass 12,000 Da)
required to associate with the template.
The enzyme has a very active 5'-3' polymerase activity that can
incorporate 300 nucleotides/s. It is also the most highly processive of
all the DNA polymerases in that it can extend a primer for several
kilobases without the enzyme dissociating from the template. The
enzyme is also very tolerant of nucleotide analogs, which renders it
very useful for DNA sequencing and oligolabeling techniques.
T7 DNA polymerase lacks a 5'-3' exonuclease activity (16), but like
T4 DNA polymerase, it has a potent 3'-5' exonuclease. In this case, the
activity is 1000-fold that exhibited by Klenow fragment. The 3'-5'
exonuclease activity can be inactivated by incubation with a reducing
agent, oxygen, and low concentrations of Fe E+. This modification does
not affect the polymerization ability of the enzyme (17). Chemical
modification removes 99% of the exonuclease activity, and total re-
moval has been achieved by genetic engineering. These modified
versions of T7 DNA polymerase are readily available commercially.