Klenow fragment pdf12/3/2023 ![]() ![]() Preparation of radioactive DNA probes: Examine each of the reactions depicted above. ![]() This reaction is more efficienty conducted with T4 DNA polymerase, which has much more potent exonuclease activity. Removal of nucleotides from the 3' ends will continue, but, in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends. The 3' -> 5' exonuclease activity of Klenow will digest away the protruding overhang. This reaction is conceptually identical to the one described above, but with a huge primer and a very short segment of single-stranded template.ĭigesting away protruding 3' overhangs: This is another method for producing blunt ends on DNA, in this with ends generated from restriction enzymes that cleave to produce 3' overhangs. One item of some significance in the above reaction is that as Klenow proceeds, it can displace primers downstream and continue synthesizing new DNA.įilling in recessed 3' ends of DNA fragments: A "fill-in" reaction is used to create blunt ends on fragments created by cleavage with restriction enzymes that leave 5' overhangs. The reaction proceeds are depicted below: To use Klenow to synthesize a complementary strand of DNA, one simply mixes single-stranded template (usually denatured double-standed DNA), primers and the enzyme in the presence of an appropriate buffer (most restriction enzyme buffers work well). The oligonucleotides must be complemenary to some section of template DNA. The primers used for most in vitro polymerization reactions are single-stranded DNAs, typically 6 to 20 bases in length, called oligonucleotides. Performing that task in the lab is integral to such processes as synthesizing the second strand DNA in cDNA cloning and generating radioactive probes for hybridization reactions.ĭNA polymerases require a primer to provide a free 3' hydroxyl group for initiation of synthesis. Synthesis of double-stranded DNA from single-stranded templates: The function of DNA polymerases is to synthesize complementary strands during DNA replication. Klenow fragment is useful for several tasks: The enzyme you purchase is almost certainly produced in this manner. In addition to generating Klenow fragment by proteolysis, it can be expressed in bacteria from a truncated form of the DNA polymerase I gene. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3' -> 5' exonuclease activities, and is widely used in molecular biology. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment. However, this pesky enzymatic activity can readily be removed from the holoenzyme. coli's DNA polymerase I makes it unsuitable for many applications. ![]()
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