RECOMBINANT TECHNIQUES.pptx, Dr Madhushree Pahari

Recombinant techniques , restriction endonuclease, cloning vector,plasmid, transfection, DNA isolation, agarose gel electrophoresis, southern blotting technique

1. RECOMBINANT TECHNIQUES Presented by Dr. Madhushree Pahari 2 nd year PGT IPGME&R MODERATOR Prof(Dr.) Mousumi Mukhopadhyay HOD,Dept.Of Biochemistry IPGME&R and SSKMH

2. What is recombinant technique? Techniques by which genetic recombination is carried away in vitro. It entails breakage and rejoining of DNA molecules from different organisms and production and isolation of modified DNA via artificial means.

3. DISCOVERY

4. Goal of recombinant techniques Isolate and characterize a gene Artificially synthesize new gene Alternating genome of an organism

5. BASIC PRINCIPLES OF RECOMBINANT DNA TECHNIQUE To produce DNA fragments using restriction enzymes. Joining DNA fragments to other vector DNA molecules by various techniques of ligation Cloning which is inserting resultant recombinant DNA molecule into host cell where they self replicate to produce multiple identical copies per cell of inserted DNA molecule

6. PROCEDURES OF MAKING RECOMBINANT DNA(rDNA) ISOLATING desired DNA CUTTING DNA with RESTRICTION ENZYME AMPLIFICATION with PCR SEPERATION of DNA BY GEL ELECTROPHORESIS LIGATION OF DNA MOLECULES INSERTING rDNA into host cell ISOLATION of recombinant cells

7. Tools used in recombinant techniques ENZYMES : Restriction endonuclease ,DNA ligase ,DNA polymerase,alkaline phosphatase,reverse transcriptase VECTOR :Plasmid,Bacteriophage,Cosmid,YAC,BAC,MAC Host :prokaryotic>E coli,B subtilis,Eukaryote>fungi :saccharomyces cerevisiae,plant cell,mammalian cell

8. RESTRICTION ENDONUCLEASE Bacteria use restriction enzymes to defend against bacteriophages Restriction enzymes were named for their ability to restrict , or limit, the number of strains of bacteriophage that can infect a bacterium. HindII was the first restriction enzyme to be isolated. This enzyme was first isolated from Haemophilus influenzae Rd strain II.

9. RESTRICTION MODIFICATION SYSTEM (Restriction endonuclease + methylase) is termed as a restriction modification system, found in bacteria. A bacterium is immune to its own restriction enzymes, even if it has the target sequences because the bacterial restriction sites are highly methylated , making them unrecognizable to the restriction enzyme.

10. Restriction Modification SYSTEM

11. RESTRICTION ENDONUCLEASE ENDONUCLEASE of prokaryotes also known as molecular scissor that catalyzes the degradation of foreign DNA of a different bacteria or a phage. Restriction enzyme recognise specific base sequences in DNA which is generally palindromes, 4 -8 base pair in length known as recognition or restriction site THREE MAJOR TYPES TYPE I AND TYPE III: recognise restriction site but type I make cut 1000 bp and type III make cut 25 bp away from recognition site

12. Type II restriction endonuclease Recognize restriction site in DNA and make double-stranded cuts within the specific base sequence in DNA. These cuts based on restriction enzymes are of two types A)Blunt End and B)Sticky End Blunt End: breaks occur at the centre of symmetry within recognition site Sticky End: breaks occur equidistant from and on opposite sides of the centre of symmetry within recognition site .

13. Sticky end ,blunt end Sticky end Ligation is efficient Could base pair with themselves to form dimers and new DNA molecule remain blunt ended Blunt end Ligation is less efficient Need high concentration of DNA and ligase

14. STICKY END,BLUNT END

15. NUCLEIC ACID LINKERS :treatment of blunt end Chemically synthesized double stranded DNA oligonucleotides containing one or more restriction sites for restriction enzymes :EcoRI,Hind III,Bam HI etc. Linkers are ligated to blunt end with DNA ligase enzyme ,then it is digested with specific restriction enzymes to produce sticky ends as ligation of blunt end DNA fragments with cloning plasmid is not efficient as sticky end DNA fragments

16. Treatment of blunt end

17. HOMOPOLYMER TAILING Sticky end can be produced on a blunt ended DNA molecule Terminal deoxynucleotidyl transferase enzyme is used to add homopolymer tail adds a series of nucleotides on the 3'-OH end of a double-stranded DNA molecule. In a homopolymer all the subunits are same ex;polydeoxyguanosine poly d(G)

18. Homopolymer tailing

19. CLONING VECTOR   Vectors are used as carriers to deliver the desired foreign DNA into a host cell All have at least one unique cloning site ,a sequence that can be cut by a restriction endonuclease to allow site specific insertion of foreign DNA Contain sequence that allow them to be replicated autonomously within a compatible host

20. Plasmid Small circular extra chromosomal double stranded DNA molecule Can replicate independently Found in almost in all bacteria and some fungi,protozoa,Plants and animals

21. Plasmid replication

22. PLASMID MAP Plasmid maps  are graphical representation of  plasmids , that show the locations of three key parts Origin of replication Selectable marker gene Cloning site:restriction site where DNA is inserted

23. A schematic representation of the pBR322 vector with restriction sites

24. Vectors:insert size

25. YAC:YEAST ARTIFICIAL CHROMOSOME Genetically engineered DNA molecule Used to clone DNA sequences in yeast cell Mimics yeast chromosome Linear molecule composed of a centromere telomeres,ARS(AUTONOMUS REPLICATING SEQUENCE) and selectable markers Capable of replicating in common bacterial host E.coli as well as in yeast s.cerevisiae

26. YAC:high capacity cloning vector

27. vectors Bacteriophage:have the ability to replicate within the bacterial cells and they provide the origin of replication Cosmid:hybrid plasmid that contains a Lambda phage cos sequence.

28. TRANSFECTION Transfection  is the process of deliberately introducing purified nucleic acids into eukaryote Some of the commonly used transfection techniques include calcium phosphate precipitation, lipofection, electroporation, and viral delivery . Transfection can either be transient or stable . Transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome. Stable transfection, foreign DNA is either integrated into the cellular genome or maintained as an extrachromosomal replicating chromosome.

29. TRANSFECTION Stable transfection Transient transfection

30. ISOLATION OF DNA ENZYMES USED IN DNA EXTRACTION LYSOZYME:lysis activity against gram negative bacterial cell wall Achromopeptidase :lysis activity against gram positive bacterial cell wall RNase A:degrade single stranded RNA Proteinase K:digestion of protein

31. ISOLATION OF DNA Reagent used other than enzymes: SODIUM DODECYL SULFATE(SDS):solubilization cell membrane lipid PHENOL:CHLOROFORM:ISOAMYL ALCOHOL SOLUTION(PCI 25:24:1):separation of DNA from other cellular components Ethanol 100%:precipitate DNA from solution Tris EDTA buffer: used to store purified DNA Chelating agent EDTA:protects DNA from nuclease enymes

32. ISOLATION OF BACTERIAL DNA BASIC STEPS A) LYSIS :1)done by gentlest possible method in presence of Chelating agent EDTA to prevent DNA fragmentation by inhibiting Dnase activity 2)Treated with detergent sodium dodecyl sulfate and lysozyme,achromopepidase B) ENZYME TREATMENT :homogenized cells are treated with a)RNase A b)proteinase K for degradation of RNA and PROTEIN C) CENTRIFUGATION : centrifugation of buffer lysed cell solution1)pellet containing cell debris 2)supernatant containing intracellular components

33. ISOLATION OF BACTERIAL DNA D) PHENOL CHLOROFORM EXTRACTION :supernatant treated with equal amount of phenol chloroform isoamyl alcohol solution, centrifugation of mixture to get supernatant aqueous layer with DNA molecules E) ALCOHOL PRECIPITATION : done adding 70% to 100% ethanol to aqueous DNA solution F) REDISSOLVE DNA :Tris-EDTA buffer used to store purified DNA

34. Isolation of Bacterial DNA

35. SEPERATION OF DNA Gel electrophoresis is a technique used to separate DNA fragments according to their size and charge. Principle :DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode. Agarose is appropriate for separating DNA fragments ranging in size from a few hundred base pairs to large DNA FRAGMENTS about 20 kb. Ethidium Bromide (EtBr) is sometimes added to running buffer during the separation of DNA fragments by agarose gel electrophoresis, EtBr binds to the DNA and allows it to visualize the DNA under ultraviolet (UV) light.

36. Agarose gel electrophoresis Gels for DNA separation are made out of a polysaccharide called agarose Agarose heated in a buffer and allowed to cool to prepare gel Buffers in gel electrophoresis are used to provide ions that carry a current necessary for passage of electricity through gel and to maintain the pH at a relatively constant value,buffer also keep gel from melting. Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE) buffers used. TAE has the lowest buffering capacity, but it provides the best resolution for larger DNA.

37. AGAROSE GEL ELECTROPHORESIS

38. DETECTION OF SPECIFIC DNA SEQUENCE Southern blotting is a technique that allows the detection of a specific region of DNA, The technique was named after its inventor, Edwin Southern. BASIC STEPS OF SOUTHERN BLOT ANALYSIS : Step 1 DNA purification and RESTRICTION digestion. Step 2 Gel electrophoresis. Step 3 Denaturation with alkali solution. Step 4 Blotting. Step 5 Hybridization with labeled probe and washing. Step 6 Detection.

39. Southern blotting technique

40. Molecular Cloning Process Of creating clones of organism or copies of cell or DNA fragments Cloning of any DNA fragment essentially involves four steps fragmentation - breaking apart a strand of DNA ligation – gluing together pieces of DNA in a desired sequence transfection – inserting the newly formed pieces of DNA into cells screening/selection – selecting out the cells that were successfully transfected with the new DNA

41. Molecular cloning

42. REFERENCES Wilson and Walker’s principles and techniques of Biochemistry and Molecular biology;8 th edition Lehninger PRINCIPLES of BIOCHEMISTRY;7th edition

43. THANK YOU