![]() ![]() ![]() ![]() This 3 to 1 ratio of DNA insert to vector is desirable, because it ups the probability of the insert being ligated into vector versus vector ligating itself. Based on the calculated molecular concentration of vector and insert, a 3 to 1 ratio of insert to vector is calculated, to determine the volume of vector and insert used in the reaction. Following gel-purification of digested vector and insert, DNA concentrations are measured a spectrophotometer to determine the concentration of the purified vector and insert.įrom this concentration, the number of molecules of insert or vector in 1 µl can be determined based on the average molecular weight for a DNA base pair and the number of base pairs in each fragment. Alkaline phosphatase treatment of vector DNA post-digestion removes 5’phosphates on both ends and prevents this undesirable outcome.Īs we mentioned previously, vector and insert DNAs are digested with endonucleases prior to beginning a ligation. When the goal is to insert a gene into a plasmid, resealing of vector DNA, called self-ligation, is a common undesirable outcome for a ligation reaction. Klenow possesses 3’ to 5’ exonuclease activity that chews up 3’ overhangs and polymerase activity that blunts 5’overhangs by extending the 3’ end of the complementary strand. However, the Klenow fragment, the product of DNA polymerase 1, digested with subtilisin can convert sticky ends to blunt ends. Sticky and blunt ends cannot, under normal circumstances, be ligated together. Because blunt end ligations don’t have any complimentary base pairing, the ligation is less efficient and more difficult for the enzyme to join the ends. Ligating sticky ends is advantageous, because the complimentary overhanging base pairs stabilize the reaction. These nicks can resemble single strand breaks producing 3’ and 5’ overhangs, called sticky ends or double strand breaks with no overhangs, called blunt-ends. In the laboratory, DNA ligases is routinely used in molecular cloning - a process that joins endonuclease-digested DNA fragments, or inserts, with an endonuclease-digested vector, such as a plasmid, so that the fragment can be introduced into host cells and then replicated.Įndonuclease digestions involve the use of restriction endonucleases, or restriction enzymes, which create nicks at specific stretches of DNA. Ligation occurs as part of normal cellular processes, such as DNA replication, to repair single and double strand DNA breaks. In the cell, DNA ligases are enzymes that identify and seal breaks in DNA by catalyzing the formation of phosphodiester bonds between the 3’-hydroxyl and 5’-phosphate groups of the DNA backbone. Molecular tools that assist with ligations like the Klenow Fragment and shrimp alkaline phosphatase (SAP) are mentioned, and applications, such as proximity ligations and the addition of linkers to fragments for sequencing are also presented. Critical aspects of ligation reactions are discussed, such as how the length of a sticky end overhang affects the reaction temperature and how the ratio of DNA insert to vector should be tailored to prevent self-ligation. The basic principle of ligation is described as well as a step-by-step procedure for setting up a generalized ligation reaction. This video provides an introduction to DNA ligation. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. In the cell, ligases repair single and double strand breaks that occur during DNA replication. An enzyme known as a ligase catalyzes the ligation reaction. In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. ![]()
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