DNA Polymerase 253.3.2.3. PROTOCOL- Dilute 1 ~tg of target DNA to 44 laL with distilled water.
- Add 5 IxL of reaction buffer.
- Optionally, add NaCI to a concentration of 0-50 mM, reducing the vol-
ume of water use in step 1 pro rata. - Add 1 IxL (0.5 U) of Klenow fragment.
- Incubate at 25°C for 15 min.
- Terminate the reaction by incubating at 70°C for 10 min.
- T4 DNA Polymerase
T4 DNA polymerase is produced by the E. coli bacterium when
infected with bacteriophage T4.4.1. Enzyme Data
It is a monomeric enzyme ofmol mass 114,000 Da, that will sequen-
tially add mononucleotide units to a DNA primer by means of its 5'-3'
polymerase activity. The enzyme cannot utilize nicked DNA as a
substrate, since its lack of a 5'-3' exonuclease activity (13) prevents it
from displacing the existing downstream DNA strand. The enzyme
has a highly active 3'-5' proofreading exonuclease activity, 200-fold
more active than that of Klenow fragment, which again is more active
against single- than double-stranded DNA.4.2. Uses ofT4 DNA Polymerase
T4 DNA polymerase is used to label 3' termini in the same manner
as Klenow fragment. The reaction can be performed at 37°C or at room
temperature, but incubation at 12°C maximizes the polymerase activ-
ity over that of the exonuclease.
The exonucleolysis/in-filling process can of course be performed in
the absence of labeled deoxynucleotide triphosphate to convert cohe-
sive or "ragged" termini to "polished" flush ends suitable for cloning.
A further use of T4 DNA polymerase is in the process of"gap-filling"
during site-directed mutagenesis. This entails the conversion of a
partially single-stranded molecule to a fully double-stranded entity.
For this application, it is usual to include the T4 gene 32 protein, which
stimulates DNA synthesis (14).