![]() ![]() Thus, the Klenow fragment is very useful in double-stranded DNA synthesis (second strand cDNA synthesis), blunting by filling of 3´ ends (after restriction digestion or just filling in of large gaps), primer labeling (radiolabeled nucleotides onto 3’ ends), and DNA sequencing experiments. The small fragment contains the 5´ - 3´exonuclease activity while the large fragment or Klenow fragment retains the 5´ - 3´ polymerase and 3´ - 5´ proofreading activities. Fortunately, researchers were able to separate these different functions by separating Pol I into two fragments. The exonuclease activity of Pol I limits its use in molecular experiments because the 5´ - 3´ exonuclease activity might interfere with generating single-stranded DNA for downstream applications. Proofreading (3´ - 5´ exonuclease) actvity Polymerizes 1000 nucleotides per minute. ![]() 3´ - 5´ exonuclease activity (proofreading).Pol I’s molecular weight is 109 kDa and its optimal temperature is 37☌ and can polymerize 1000 nucleotides per minute. Pol I is also commonly used for 3´ DNA end removal or filling. In addition, Pol I has a 5´ - 3´ exonuclease activity, which makes this enzyme useful in excision repair upstream of polymerization, removal of RNA primer, and nick translation. On the other hand, the 3´ - 5´ exonuclease activity results in proofreading of the new DNA strand and removal of any mistakes. The DNA polymerase activity achieves replication of DNA in a 5´ - 3´ direction and requires deoxyribo-nucleotides triphosphate (dNTPs) and a primer. Pol I is made up of multiple domains that exhibit two different enzymatic activities: DNA-dependent DNA polymerase activity and exonuclease activity. Since then, many other polymerase enzymes have been discovered in bacteriophages or phages and engineered to produce amplicons for many downstream applications. The first DNA polymerase, Pol I, was discovered in the late 1950s by Arthur Kornberg and his colleagues by purifying it from E. Here, we provide you with a list of the relevant characteristics and functions of the most commonly used DNA polymerases to help you choose the right enzyme for your experiment.įor a quick comparison, scroll down or click here to view our chart of relevant characteristics of the polymerases discussed. There are many different polymerase enzymes available that exhibit different amplification abilities as well as various levels of fidelity (error rate) and processivity (number of bases a polymerase can add before disassociating from its substrate – template strand). Aside from PCR, polymerases are used in a myriad of applications that require polymerases with specific abilities. DNA polymerase is an important and necessary reagent used every day in thousands of molecular biology laboratories around the ![]()
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