Recombinant DNA technology and Molecular Biology Techniques

1. Recombinant DNA technology and Molecular Biology Techniques Dr.S.Chakravarty MD

2. Learning objectives • Describe the Concept of cloning and its applications • List the steps in PCR and its clinical applications • Explain the Principles of RFLP • Discuss the Concept of Gene therapy and its clinical applications • Explain the principles of Gel electrophoresis and its clinical applications • Discuss the various types of blotting techniques and enlist the uses

3. PCR – POLYMERASE CHAIN REACTION • Karry Mullis Nobel Prize 1993

4. Yellowstone National park Enzyme taqpoloymerase obtained from bacteria Thermusacquaticus found in Hot Springs

5. Uses of PCR • DIAGNOSIS OF BACTERIAL AND VIRAL DISEASES- even a single bacillus can be identified. • MEDICOLEGAL CASES • Diagnosis of GENETIC DISORDERS • Detection of Cancer • PALEONTOLOGICAL STUDIES

6. Polymerase Chain Reaction Polymerase Chain Reaction (PCR): Produces virtually unlimited copies of a very small DNA sample • Requirements: • Taq polymerase (DNA polymerase) • d-NTPs • Specific primers • Buffer • Magnesium chloride • DNA template

7. Polymerase Chain Reaction • Four steps of a PCR cycle • Template strand separation (Denaturation) • The test tube is heated to 90-95oC to cause the double stranded template DNA to separate into single strands… • Binding of the primers (Annealing) • The temperature is lowered to 50oC to allow the primer DNA segments to bind to the targeted gene sequences through hydrogen bonding

8. Polymerase Chain Reaction • Four steps of a PCR cycle • New DNA synthesis at targeted sequences • The temperature is raised to 70-72oC where the heat-stable DNA polymerase synthesizes new DNA of the sequences targeted by the primers… • Repetition of the cycle • The cycle is repeated automatically (by a thermocycler machine) for 20-30 cycles, producing up to 1 billion copies of the original targeted DNA sequence

9. Cycle of PCR

12. The Reaction PCR tube THERMOCYCLER

13. Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

17. Uses of PCR • DNA cloning • Gene sequencing • Diagnosis of hereditary disease – along with RFLP • Genetic finger printing • Diagnosis of infectious diseases • Forensics

18. Problem with actual amplification of genes • Both introns and exons will be present • Collect m-RNA of a particular gene and then synthesize C-DNA by using reverse transcriptase

19. Gel Electrophoresis • Gel electrophoresis is a technique used to spread out different-length DNA and protein fragments in a mixture • Depends on pore size of the gel and charge on the molecule

20. RESTRICTION ENDONUCLEASES • Restriction endonucleases hydrolyze phosphodiester linkages in both the strands of DNA at highly specific sites. • They recognize palindromes in 5’  3’ direction on both the strands. • Protect bacteria from bacteriophage infection -Restricts viral replication • Bacterium protects it’s own DNA by methylating those specific sequence motifs.The restriction endonuclease and the corresponding methylase are sometimes referred to as a restriction-modification system. • Some enzymes generate “blunt ends” (cut in middle) • Others generate “sticky ends” (staggered cuts) - H-bonding possible with complementary tails - DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones A Santa lived as a devil at NASA Live on time, emit no evil

21. RESTRICTION ENDONUCLEASES

22. Restriction endonucleases • Other enzymes yield blunt ends on cleavage Eg: Hae III HaeIII 5’---GGCC---3’ 3’---CCGG---5’ 5’---GG CC---3’ 3’---CC GG---5’ Blunt ends generated by digestion with HaeIII

23. Restriction Endonucleases • Enzymes that cleave double stranded DNA at specific sites • Cleave at Palindromes Palindromic recognition sequence for EcoRI Creates DNA fragments with sticky ends

26. Size determination • Size markers are commonly used in electrophoresis experiments • The distance migrated is related to the inverse log of the molecular weight of the DNA fragment

27. DNA Libraries Two types: • Genomic DNA libraries: Contain the entire nuclear DNA • cDNA Libraries (Expression libraries): Contain the cDNA of the mRNA that are expressed in the tissues

28. Recombinant DNA technology DNA from any Source Vector Recombinant Vector Incorporated into host bacterial cell Large quantities of the gene available for genetic diagnosis, Sequencing Host cell allowed to multiply Multiple copies of the desired gene produced Expression of the gene→ Production of desired protein

29. Vectors (Plasmids) useful in cloning • Circular DNA molecules( ds DNA ) • Contain genes for antibiotic resistance • Have many palindrome sites recognized by restriction endonucleases • CAN ACCEPT 6-10 Kbp long foreign DNA • Other vectors useful in recombinant DNA technology are: • Viral chromosome (Phage) • Cosmids • Bacterial artificial chromosome (BAC) • Yeast artificial chromosome (YAC)

30. Recombination of DNA

32. The sticky ends can reconnect !! How to overcome this problem?

33. ANSWER :- HOMOPOLYMER TAILING :- a segment of poly dG is added to one strand at 3’ end of one DNA a segment of poly dC is added to 3’end of vector DNA 5’--------------------GGGG3’ 3’GGGG-------------------5’ FOREIGN DNA 5’-------------------CCCC3’ 3’CCCC------------------5’ HOST DNA

34. DNA LIGASE • The study of DNA replication and repair processes led to the discovery of the DNA-joining enzyme called DNA ligase. • DNA ligases catalyze formation of a phosphodiester bond between the 5′-phosphate of a nucleotide on one fragment of DNA and the 3′-hydroxyl of another. • DNA ligase is required to seal the gaps, covalently bonding the two strands and regenerating a circular molecule. • The DNA ligase most widely used in the lab is derived from the bacteriophage T4.

35. How to check correct insertion into plasmid gene ? A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. REMEMBER – PLASMIDS carry genes for antibiotic resistance. This insertion disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, cells carrying the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert.

36. Production of Recombinant proteins Proteins produced by recombinant DNA technology are: • Human Insulin (Diabetes mellitus) • HBsAg (Vaccine for Hepatitis) • factor VIII (Hemophilia)

37. Genomic library Chromosomal DNA Enzymes required: Restriction endonuclease, DNA ligase Contain introns Useful for DNA sequencing (Human genome project) cDNA library cDNA synthesized from mRNA Enzymes required: Reverse transcriptase, DNA polymerase, DNA ligase Contain only exons Useful in producing recombinant proteins, gene therapy, constructing transgenic animals Comparison of genomic library and cDNA library

38. Blotting techniques • Southern blot– Analysis of specific DNA fragment of interest • Northern blot – Analysis of specific RNA of interest. • Western blot – Analysis of specific protein of interest. • Southwestern blotting – DNA protein interactions • Eastern blotting – post translational modifications of proteins

40. Southern blot • Extraction of DNA • Cleavage of the DNA using Restriction enzymes • Gel electrophoresis – separation by size • Denaturation and Blotting on nitrocellulose paper • Gene of interest is identified by probes – Radioactive

41. Southern Blot

42. Southern blot analysis can be used to detect RFLPs: DNA is digested with restriction enzyme and subject to gel electrophoresis. DNA is transferred to a nitrocellulose membrane and hybridized to a labeled DNA probe Membrane is exposed to X-ray film and DNA fragments analyzed.

43. Northern blotting • Semi quantitative procedure helpful in determining which tissues express the gene for a specific protein. • Measures amount of RNA in the sample

44. Western blotting • Diagnostic test for HIV • Diagnosis of hepatitis B infections • Detecting various proteins of interest

45. South western blotting • Used to detect DNA protein interactions • Protein of interest is extracted from the nucleus, subjected to electrophoresis, transferred to a filter and exposed to labelled DNA.

46. Gene therapy • Gene therapy is a technique for correcting defective genes responsible for disease development. • Single gene defects • Replacement therapy- extra copy, not true replacement (except through single cell homologous recombination methods) • The gene is not transferred to the offspring • Vectors useful in gene transfer: • Retrovirus • Adenovirus • Liposome (blood brain barrier)

47. ---LTR--------gag----pol----env--------LTR RETROVIRAL RNA DELETE gag,pol and env genes ---LTR-------- -------LTR Insert HUMAN DNA (Cassette containing Human gene ) ---LTR-------- --human gene -------LTR (Put viral DNA in packaging cells to get protein coat ) INSERT INTO HUMAN HOST CELL ---LTR-------- --human gene -------LTR

48. ---LTR-------- --human gene -------LTR Reverse transcriptase Human DNA is synthesized and it integrates into HOST CELL DNA INFUSE THESE MODIFIED CELLS INTO THE PATIENT !!

49. Ex vivo gene therapy Cells from the patient are removed and grown in culture Cells are infected with the modified vector Gene is incorporated into cells Modified cells are infused back into patient In vivo gene therapy Modified vector is injected into patient (contains the therapeutic gene) Vector infects cells and delivers the gene into cells Cell specific delivery of gene is difficult Gene therapy

50. The first approved gene therapy case in the United States took place on September 14, 1990, at the National Institute of Health. It was performed on a four year old girl named Ashanti DeSilva. It was a treatment for a genetic defect that left her with ADA-SCID, a severe immune system deficiency. The effects were only temporary, but successful.[19]