9/12/2023 0 Comments Klenow fragment ligation![]() Preparation of the InsertĪn Insert is any desired fragment of DNA that you want to clone into a vector/plasmid. Selection of transformants and other downstream processes.Removal of the terminal phosphate of the linearized vector (phosphatase reaction).This also increases screening efforts to get the clone with the desired orientation.īlunt-end cloning involves the following steps. It is challenging as the probability of generating a clone with multiple inserts with the right orientation is low.In certain instances where the insert concentration is high, a ligated vector can have more than one insert.Additional steps are needed to avoid self-ligation (discussed below). Blunt-end cloning is prone to vector self-ligation.This results in a little chance for a stable association between the insert and the vector, resulting in a lower recombination efficiency (10-100x) than sticky-end cloning. In blunt-end cloning, both inserts and vectors do not have complementary 3’ or 5’ overhangs.Non-directional cloning generates only 50% of inserts with the proper orientation.It is used for sub-cloning, sequencing, building libraries of DNA molecules, and expressing coding and non-coding RNA.It does not require a complementary sequence between the insert and the This property makes it a truly universal cloning method. Blunt-end cloning has an advantage over other cloning methods (TA-cloning or traditional restriction enzyme-based sticky-end cloning).Blunt-end cloning involves simple design of the primers (without any extra bases at the 5’ end of the primer).The T4 DNA ligase utilizes ATP to make a phosphodiester bond between the 3’ hydroxyl group of one DNA strand and the 5’ phosphate group of another DNA strand.Ī detailed mechanism of ligation is discussed here. During the transient association of the ends of the vector and the insert, the DNA ligase enzyme seals the joints. The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray, and also in dA and dT tailing, an important step in the process of ligating DNA adapters to DNA fragments, frequently used in preparing DNA libraries for Next-Gen sequencing.In blunt-end cloning, both the vector and the insert contain blunt-ends. This form of the enzyme is called the exo- Klenow fragment. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5'). This problem can be overcome by introducing mutations in the gene that encodes Klenow. Just as the 5' → 3' exonuclease activity of DNA polymerase I from E.coli can be undesirable, the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain applications. The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process, before being replaced by thermostable enzymes such as Taq polymerase. Filling in receded 3' ends of DNA fragments to make 5' overhang blunt.Synthesis of double-stranded DNA from single-stranded templates.The Klenow fragment is extremely useful for research-based tasks such as: coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).īecause the 5' → 3' exonuclease activity of DNA polymerase I from E. coli is cleaved by subtilisin retains the 5' → 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. The other smaller fragment formed when DNA polymerase I from E. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity. coli is enzymatically cleaved by the protease subtilisin. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).
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