20 Maundersin this manner. DNA polymerase I is the only common DNA poly-
merase that can carry out this coupled reaction by virtue of its 5'-3'
exonuclease function. A typical nick translation protocol is given in
Section 2.3.1.
Disadvantages of nick translation with regard to other methods of
probe labeling include the facts that the specific activity that may be
achieved is not high, and that at temperatures above 20°C artifactual
sequences ar e produced, such as "snapback DNA," where the newly
synthesized strand disassociates from the template and is then itself
copied to form inverted DNA sequences.
Another use of DNA polymerase I is in repair synthesis, where the
enzyme is used to "fill in" protruding 5' termini. End-labeling can also
be achieved by removal of 3' protruding termini by the 3'-5' exonu-
clease activity and then subsequently filling in the 3' recess so formed.
DNA polymerase I can also be used in eDNA synthesis, where it is
employed in the synthesis of the second DNA strand. The enzyme has
an inherent RNase H activity that is of value here. However, cDNA
synthesis is largely performed by DNA polymerase I large (Klenow)
fragment or reverse transcriptase.
2.3. Experimental Procedures
DNA polymerase I is best stored in 50 mM potassium phosphate, pH
7.0, containing 250 ktM DTT and 50% glycerol. At -20°C, it is stable
for over 6 mo.
2.3.1. Protocol for Nick Translation with DNA Polymerase I2.3.1.1. MATERIALS REQUIRED
- Probe DNA.
- Nick translation buffer:
- 500 mM Tris-HC1, pH 7.8
- 50 mMMgC12
- 100 mM ~-Mercaptoethanol
- 100 ktg/mL Bovine serum albumin
- 200 jaM dGTP.
- 200 ~/dCTP.
- 200 pM dTTP.
- 0.1 lag/mL DNase I (prepare by serial dilution of 1 mg/mL stock).
- DNA polymerase I (diluted to 2 U/laL in storage buffer).