DNA Polymerase 23
the labeled probe produced is strand-specific, and there is no produc-
tion of hairpin-like artifacts.
This reaction can of course be performed with unlabeled deoxy-
nucleotides to produce flush or modified termini for subsequent clon-
ing reactions. The fill-in reaction (see Section 3.3.2.) can be controlled
by the withholding of one particular deoxynucleotide to generate novel
cohesive ends. Excess Klenow fragment will also yield only partially
filled termini because of the 3'-5' exonuclease activity.
Klenow fragment is also used for elongating oligomeric primers in
site-directed mutagenesis to produce double-stranded DNA. How-
ever, the enzyme tends to displace mismatched oligomers and so remove
the intended mutation. T4 DNApolymerase is perhaps more useful in
this context.
The enzyme can also be used for the synthesis of the second DNA
strand during cDNA synthesis (11,12). Finally, Klenow fragment was
originally used in the polymerase chain reaction, before the introduction
of thermostable DNA polymerases permitted the process to be automated.
- Experimental Procedures
Klenow fragment has similar physical properties to Pol I and should
be stored under similar conditions. The following sections describe
experimental protocols for two techniques utilizing Klenow fragment:
radiolabeling DNA by extension of random primers and in-filling of
cohesive termini.
3.3.1. Protocol for Labeling with Random Hexameric Primers
3.3.1.1. ~/[ATERIALS REQUIRED
- Experimental Procedures
- Probe DNA.
- Reaction buffer:
- 450 mM HEPES, pH 6.6
- 50 mM MgCI 2
- 10 mM Dithiothreitol
- 2 mg/mL Bovine serum albumin
- 2.5 mM dGTP
- 2.5 mM dCTP
- 2.5 mM dTTP
- Klenow fragment (diluted to 2 U/gL in storage buffer).
- ct-32P dATP (10 mCi/mL, 3000 Ci/mmol).
- Sterile distilled water.