RECOMBINANT DNA TECHNOLOGY

RECOMBINANT DNA TECHNOLOGY

1. Recombinant DNA technology Gandham.Rajeev Email:[email protected]

2. It occurs in following s tages Generation of DNA fragments & selection of the desired piece of DNA (e.g. a human gene). Insertion of the selected DNA into a cloning vector (e.g. a plasmid) to create a recombinant DNA or chimeric DNA. Recombinant DNA technology

3. Introduction of the recombinant vectors into host cells (e.g. bacteria). Multiplication & selection of clones containing the recombinant molecules. Expression of the gene to produce the desired product.

4. rDNA technology

5. Molecular tools of genetic engineering. Host cells-the factories of cloning. Vectors-the cloning vehicles. Methods of gene transfer. Gene cloning strategies. Steps

6. Restriction endonucleases - DNA cutting enzymes: Restriction endonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the bacterial enzymes that can cut/split DNA (from any source) at specific sites. Molecular tools of genetic engineering

7. They were first discovered in E.coli restricting the replication of bacteriophages, by cutting the viral DNA (The host E. coli DNA is protected from cleavage by addition of methyl groups). Thus, the enzymes that restrict the viral replication are known as restriction enzymes or restriction endonucleases.

8. Recognition sequence is the site where the DNA is cut by a restriction endonuclease . Restriction endonucleases can specifically recognize DNA with a particular sequence of 4-8 nucleotides & cleave . Cleavage patterns: Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within recognition sequence. Recognition sequences

9. The cut DNA fragments by restriction endonucleases may have mostly sticky ends (cohesive ends) or blunt ends. DNA fragments with sticky ends are useful for recombinant DNA experiments. This is because the single-stranded sticky DNA ends can easily pair with any other DNA fragment having complementary sticky ends.

10. The cut DNA fragments are covalently joined together by DNA ligases . These enzymes were originally isolated from viruses. They also occur in E.coli & eukaryotic cells. DNA ligases actively participate in cellular DNA repair process. DNA ligases

11. The hosts are the living systems or cells in which the carrier of recombinant DNA molecule or vector can be propagated. There are different types of host cells-prokaryotic (bacteria) & eukaryotic (fungi, animals & plants). Host cells – the factories of cloning

12. Host cells, besides effectively incorporating the vector's genetic material, must be conveniently cultivated in the laboratory to collect the products. Microorganisms are preferred as host cells, since they multiply faster compared to cells of higher organisms (plants or animals).

13. Escherichia coli: Escherichia coli was the first organism used in the DNA technology & continues to be the host of choice by many workers. The major drawback is that E. coli (or even other prokaryotic organisms) cannot perform post-translational modifications. Bacillus subtilis as an alternative to E.coli . Prokaryotic hosts

14. The most commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae . Certain complex proteins which cannot be synthesized by bacteria can be produced by mammalian cells e.g. tissue plasminogen activator. The mammalian cells possess the machinery to modify the protein to the active form (post-translational modifications). Eukaryotic hosts

15. Vectors are the DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self-replicating in an appropriate host cell. The most important vectors are plasmids, bacteriophages, cosmids & artificial chromosome vectors. Vectors – The cloning vehicles

16. Plasmids are extrachromosomal , double-stranded, circular, self-replicating DNA molecules. Almost all bacteria have plasmids. Size of plasmids varies from 1 to 500 kb. Plasmids contribute to about 0.5 to 5.0% of total DNA of bacteria. Plasmid

17. Genetic map of plasmid cloning vector pBR322

18. pBR322 - the most common plasmid vector pBR322 has a DNA sequence of 4,361 bp . It carries genes resistance for ampicillin (Amp1) & tetracycline (Tel1) that serve as markers for the identification of clones carrying plasmids. The plasmid has unique recognition sites for the action of restriction endonucleases - EcoRl , Hindlll , BamHl , Sall & Pstll

19. The other plasmids employed as cloning vectors include pUC19 (2,686 bp , with ampicillin resistance gene) & derivatives of pBR322-pBR325, pBR328 & pBR329. Other plasmid cloning vectors

20. Bacteriophages or phages are the viruses that replicate within the bacteria. In case of certain phages, their DNA gets incorporated into the bacterial chromosome & remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. Bacteriophages

21. Phages can take up larger DNA segments than plasmids. Phage vectors are preferred for working with genomes of human cells. The most commonly used phages are bacteriophage λ (phage λ ) & bacteriophage (phage M13). Advantage of phages

22. Cosmids are victors possessing the characteristics of both plasmid & phage λ . Cosmids can be constructed by adding a fragment of phage λ DNA including Cos site, to plas mids . A foreign DNA (about 40 kb) can be inserted into cosmid DNA . The recombinant DNA, formed can be packed as phages & injected into E.coli . Inside host cell, cosmids behave like plasmids & replicate & can carry larger fragments of foreign DNA Cosmids

23. Human artificial chromosome (HAC): Artificial chromosome is a synthetically produced vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a self-replicating microchromosome with a size ranging from 1/10 th to 1/5 th of a human chromosome. It can carry long human genes. Artificial chromosome vectors

24. Yeast artificial chromosome (YAC) is a synthetic DNA that can accept large fragments of foreign DNA (particularly human DNA). It is possible to clone large DNA pieces by using YAC. Yeast artificial chromosome (YACs)

25. Construction of BACs is based on one F-plasmid which is larger than the other plasmids used as cloning vectors. BACs can accept DNA inserts of around 300 kb. Bacterial artificial chromosome (BACs)

26. Transformation: Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli ). Uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl2 (0-5˚C) & a subsequent heat shock (37-45 ˚ C for about 90 sec). Methods of gene transfer

27. A natural microbial recombination process. During conjugation, two live bacteria (a donor & a recipient) come together, join by cytoplasmic bridges & transfer single stranded DNA (from donor to recipient). In side recipient cell, new DNA may integrate with the chromosome or may remain free. Conjugation

28. It is a technique involving electric field mediated membrane permeabiIization. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution. It is a simple & rapid technique for introducing genes into cells. Electroporation

29. Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids. Several techniques have been developed to encapsulate DNA in liposomes. The liposome mediated gene transfer is referred to as lipofection . Liposome-mediated gene transfer

30. Treatment of DNA fragment with liposomes, DNA pieces get encapsulated inside liposomes. These liposomes can adhere to cell membranes & fuse with them to transfer DNA fragments. The DNA enters the cell & to the nucleus. Positively charged liposomes efficiently complex with DNA, bind to cells & transfer DNA

31. It is possible to directly transfer the DNA into the cell nucleus. Microinjection & particle bombardment are the two techniques used for this purpose. Direct transfer of DNA

32. A clone refers to a group of organisms, cells, molecules or other objects , arising from a single individual. Gene cloning strategies

33. Gene cloning strategies Generation of DNA fragments Insertion into a cloning vector Introduction into host cells Selection or screening RE-digestion, cDNA synthesis, PCR, chemical synthesis Ligation of blunt ends, homopolymer tailing, linker molecules Transformation, transfection , tradsduction Hybridization, PCR, immunochemical methods, protein-protein interactions, functional complementation

34. Textbook of Biochemistry – U Satyanarayana References

35. Thank You