Sunteți pe pagina 1din 15

FUNDAMENTALS OF BIOTECHNOLOGY

TRANSFERRING GENE OF INTEREST INTO VECTORS

Plasmid Vectors
Plasmid vectors are small circular molecules of double stranded DNA derived from natural plasmids that occur in bacterial cells. 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.

The plasmid and the foreign DNA are cut by this restriction endonuclease (EcoRI in this example) producing intermediates with sticky and complementary ends. Those two intermediates recombine by base-pairing and are linked by the action of DNA ligase. A new plasmid containing the foreign DNA as an insert is obtained. A few mismatches occur, producing an undesirable recombinant.

When same sticky end creating enzyme used for cleavage of vector and gene of interest, then DNA ligase seals the nick between gene of interest and vector and creates recombinant vector. Whereas when blunt end creating enzyme used then recoiling become difficult. Moreover, in both cases of using sticky end and blunt end enzymes, self coiling of vector also occur in high rate rather than the recombination of vector and genes. These situations are overcome by using i) Linkers ii) Adaptors iii) Homopolymer tailing

Sticky ends increase the efficiency of ligation

Figure The different joining reactions catalysed by DNA ligase: (a) ligation of blunt-ended molecules; (b) ligation of sticky-ended molecules.

i) Linkers:
Linker molecules are used to ligate the blunt end gene of interest with cohesive end vectors. They are normally synthesized self-complementary decameric oligonucleotiedes, which contain sites for one or more restriction endonucleases which will create sticky ends. The linker can be ligated to both ends of the foreign gene to be clones, 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.

Putting sticky ends onto a blunt-ended molecule

Figure Linkers and their use: (a) the structure of a typical linker; (b) the attachment of linkers to a blunt-ended molecule.

Figure A possible problem with the use of linkers. Compare this situation with the desired result of BamHI restriction, as shown in Figure 4.21(b).

ii) Adaptors:
When linkers added to link at the end of blunt end of gene interest, then there is an possibility of joining of multiple linkers at the end. This makes some time larger genes and waste of linker molecules. This problem is overcome by using adapters. Since adapters contain only one end suitable for joining this prevents multiple coiling of adapters. Adapter is a synthetic, double stranded oligonucleotide used to attach sticky ends to a blunt ended molecule. 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. This is achieved by using alkaline phosphatases. In contrast to linkers, adapters contain preformed sticky ends and joining blunt ends. Because of lack of 5' phosphate group on sticky end prevents adapter polymer formation. 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, producing sticky ended fragment that can be inserted into an appropriate vector. Eventhough adaptors prevents polymer formation, it does not prevents self ligation or recoiling of vectors during recombination reaction. This disadvantages nature of adaptors removed by using homopolymer tailing.

Figure Adaptors and the potential problem with their use. (a) A typical adaptor. (b) Two adaptors could ligate to one another to produce a molecule similar to a linker, so that (c) after ligation of adaptors a blunt-ended molecule is still blunt-ended and the restriction step is still needed.

Figure The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5-OH terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors.

iii) Homopolymer Tailing:


A homopolymer is simply a polymer in which all the subunits are the same. Tailing involves using the enzyme terminal deoxynucleotidyl transferase, to add a series of nucleotides on to the 3'-OH termini of a double stranded DNA molecule. If this reaction is carried out in the presence of just one deoxynucleotide, then a homopolymer tail will be produced. In this method, 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. In this case recoiling of vectors are mostly prevented because vector does not contain complementary ends. The new recombinant vector can be introduced into bacterial cells that can produce many copies of the inserted DNA . This technique is called DNA cloning.

Figure Homopolymer tailing: (a) synthesis of a homopolymer tail; (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA; (c) repair of the recombinant DNA molecule.

A Useful Link For This Course:

http://biosiva.50webs.org/dnacloning.htm