FUNDAMENTALS OF BIOTECHNOLOGYTRANSFERRING GENE OF INTEREST INTO VECTORS Those two intermediates recombine by base-pairing and are linked by the action of DNA ligase. The plasmid and the foreign DNA are cut by this restriction endonuclease (EcoRI in this example) producing intermediates with sticky and complementary ends. producing an undesirable recombinant. . A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for same restriction endonuclease.Plasmid Vectors • Plasmid vectors are small circular molecules of double stranded DNA derived from natural plasmids that occur in bacterial cells. A new plasmid containing the foreign DNA as an insert is obtained. A few mismatches occur. Whereas when blunt end creating enzyme used then recoiling become difficult. then DNA ligase seals the nick between gene of interest and vector and creates recombinant vector. These situations are overcome by using • i) Linkers • ii) Adaptors • iii) Homopolymer tailing . in both cases of using sticky end and blunt end enzymes. Moreover.• When same sticky end creating enzyme used for cleavage of vector and gene of interest. self coiling of vector also occur in high rate rather than the recombination of vector and genes. (b) ligation of sticky-ended molecules.Sticky ends increase the efficiency of ligation Figure The different joining reactions catalysed by DNA ligase: (a) ligation of blunt-ended molecules. . and then treated with restriction endonuclease to produce a sticky ended fragment which can be incorporated into a vector molecule that has been cut with the same restriction endonuclease. Insertion by means of the linkers creates restriction enzyme target sites at each end of the foreign gene and so enables the foreign gene to be excised and recovered after cloning and amplification in the host bacterium.i) Linkers: Linker molecules are used to ligate the blunt end gene of interest with cohesive end vectors. The linker can be ligated to both ends of the foreign gene to be clones. . which contain sites for one or more restriction endonucleases which will create sticky ends. They are normally synthesized self-complementary decameric oligonucleotiedes. . . (b) the attachment of linkers to a blunt-ended molecule.Putting sticky ends onto a blunt-ended molecule Figure Linkers and their use: (a) the structure of a typical linker. 21(b). as shown in Figure 4. Compare this situation with the desired result of BamHI restriction. .Figure A possible problem with the use of linkers. It contain normal 5' and 3' end at blunt end and the sticky end of adapter molecule is modified in such manner that it contain OH group on both 5' and 3' ends. Adapter is a synthetic. This makes some time larger genes and waste of linker molecules. This is achieved by using alkaline phosphatases. This disadvantages nature of adaptors removed by using homopolymer tailing. Eventhough adaptors prevents polymer formation. Since adapters contain only one end suitable for joining this prevents multiple coiling of adapters. double stranded oligonucleotide used to attach sticky ends to a blunt ended molecule. it does not prevents self ligation or recoiling of vectors during recombination reaction. adapters contain preformed sticky ends and joining blunt ends.ii) Adaptors: When linkers added to link at the end of blunt end of gene interest. This problem is overcome by using adapters. Because of lack of 5' phosphate group on sticky end prevents adapter polymer formation. then there is an possibility of joining of multiple linkers at the end. . In contrast to linkers. producing sticky ended fragment that can be inserted into an appropriate vector. After the adaptors have been attached the abnormal 5'OH terminus is converted to the natural 5'P form by treatment with the enzyme polynucleotide kinase. (a) A typical adaptor. .Figure Adaptors and the potential problem with their use. so that (c) after ligation of adaptors a blunt-ended molecule is still blunt-ended and the restriction step is still needed. (b) Two adaptors could ligate to one another to produce a molecule similar to a linker. . showing the modified 5′-OH terminus. (b) conversion of blunt ends to sticky ends through the attachment of adaptors.Figure The use of adaptors: (a) the actual structure of an adaptor. gene of interest is tailed with one nucleotide and vector is tailed with an complementary base and when they are combined then only vector recombined with gene of interest. then a homopolymer tail will be produced. In this case recoiling of vectors are mostly prevented because vector does not contain complementary ends. This technique is called DNA cloning. to add a series of nucleotides on to the 3'-OH termini of a double stranded DNA molecule. . Tailing involves using the enzyme terminal deoxynucleotidyl transferase. In this method. The new recombinant vector can be introduced into bacterial cells that can produce many copies of the inserted DNA . If this reaction is carried out in the presence of just one deoxynucleotide.iii) Homopolymer Tailing: A homopolymer is simply a polymer in which all the subunits are the same. . . (c) repair of the recombinant DNA molecule.Figure Homopolymer tailing: (a) synthesis of a homopolymer tail. (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA. org/dnacloning.A Useful Link For This Course: http://biosiva.50webs.htm .