Basics of DNA Cloning-II

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Note: Before starting this lecture students should have completed Lecture 35

Sequential steps involved in DNA cloning using plasmid DNA as vector: Molecular cloning using a plasmid vector involves five major steps as shown in Fig. 1 and Fig. 2

Step 1 : Isolation of DNA (gene of interest and vector):

The first initial step in cloning a DNA fragment is to isolate foreign DNA containing gene of interest and bacterial plasmid. If the sequence of the gene of interest is known it is isolated by PCR amplification using gene specific primers which include restriction sites selected from the multiple cloning site of the plasmid selected for cloning. When the sequence of the gene is not known degenerate primers are used for PCR amplification. Most of the time people generate genomic DNA library and screen for the gene using southern hybridization technique. According to the result of southern hybridization, the DNA is sequenced and the gene was confirmed by BLAST analysis. Now the gene is amplified by PCR and cloned. There are many plasmids available commercially for cloning.

Step 2 : Treatment of plasmid and foreign DNA with the same restriction enzyme and ligation :

The gene of interest and the plasmid are modified using same restriction enzymes. Plasmid vectors are engineered to contain a specific antibiotic resistance gene and a multiple cloning site (also called the polylinker region) which contain many unique target sites for restriction endonucleases. When the circular plasmid is cut with one of the restriction enzyme whose restriction site is present in the plasmid, it results the linearization of plasmid. A fragment of DNA molecule, referred to as the "insert," is treated with the same restriction enzyme, and then can be joined to the plasmid DNA in a ligation reaction. The chance for recombinant clones in ligations of the insert to vector will not be 100% as there is more possibility of self-ligation of two ends of the plasmid. To decrease the degree of self-ligation, enzyme phosphatase is used which removes the terminal 5'-phosphate and prevents self-ligation. Another strategy to overcome self-ligation is by using two different restriction enzymes cutting sites with non- complementary sticky ends. In this way self- ligation is inhibited and also promotes correct orientation of the insert DNA within the plasmid. The ligation of the digested insert and the plasmid is performed by pooling both in a single reaction tube and adding DNA ligase enzyme which catalyses the formation of phosphodiester bond between insert and plasmid DNAs, there by forming the recombinant DNA molecule.