DNA polymerase Klenow

Another enzyme, which shares many of the properties of the DNA polymerase (Klenow sub-fragment) is the phage T4 encoded polymerase. The enzyme is a single polypeptide chain similar in size to Pol I of E. coli but which totally lacks the 5 ->3 ... 

In some circumstances the aim of the experiment will be to clone a DNA sequence which lacks cohesive ends. Such molecules may, for example, be the cDNA products from the reverse transcription of particular mRNA species and this is currently the most widely used procedure for sequencing eukaryotic mRNAs. Under controlled conditions the reverse transcriptase from Avianmyeloblas-tosis virus (AMV) will use a mRNA template to synthesize a full-length double-stranded DNA copy (cDNA) of the RNA sequence. The products of this reaction frequently lack precisely defined ends and it is necessary to trim such frayed or uneven termini before proceeding to the cloning step. Incubation with DNA polymerase (Klenow) or T4-polymerase (Section 4.2.2.) will yield molecules with clean blunt-ended termini which can be... 

Four separate reactions are set up in the tips of drawn out capillary tubes. Each reaction mixture contains the four deoxynucleotide triphosphates one of which in each case is present in limiting amounts corresponding to the dideoxynucleotide added. In the protocol described a-[32P]-dATP is used as the label but, with the appropriate changes in the compositions of the different dNTP mixes, any of the a-pP] labelled nucleotides could be used. The extension reaction is catalysed using DNA polymerase (Klenow subfragment)... 

Alternative chain extension reaction using reverse transcriptase Reverse transcriptase can be used as an effective alternative to DNA polymerase (Klenow subfragment) in the chain-termination reaction. The following protocol is taken from Smith (1980). 

Antisense RNA amphfication (aRNA) Target T4 DNA polymerase Klenow SI nuclease T7 polymerase No... 

The upstream primer (5 -CACAATTCCACACAAC) binds upstream of the insert and is extended by DNA polymerase (Klenow) away from the insert. Any labeled dNTP, as in random priming, can be used to obtain a probe with reasonably high activity. Under the experimental conditions (Brown et al., 1982 Hu and Messing, 1982), the synthesis is not allowed to reach completion to leave the insert region S5. After phenol extraction and spin chromatography on Sephadex G-50, the probe is ready for use (no heat-denaturation ). This method is extremely simple and yields strand-specific probes. 

Ribonucleotide reductase activity was assayed based on CDP reduction, using a modified method of Jong et al. (1998), with the [ CICDP reduction product determined as radioactivity incorporated into DNA in a series of two coupled reactions, catalyzed by nucleoside diphosphate kinase and DNA polymerase (Klenow fragment). A 40 pi reaction mixture contained 50 mM Hepes pH 7.2, 10 mM dithiothreitol. 

Restriction enzymes, T4-DNA ligase, DNA polymerase (Klenow fragment), T4 DNA kinase and dideoxynucleotides were from Boehringer Mannheim and used as specified by the supplier. Goat anti-rabbit IgG conjugated to alkaline phosphatase was obtained from Sigma. 

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