Recombinant DNA Technology

Recombinant DNA Technology for MBBS course

1. Recombinant DNA Technology Dr. Farhana Atia Assistant Professor Department of Biochemistry Nilphamari Medical College, Nilphamari Email: [email protected]

2. RED BIOTECHNOLOGY WHITE BIOTECHNOLOGY GREEN BIOTECHNOLOGY BLUE BIOTECHNOLOGY Medical biotechnology: manufacture pharmaceuticals, used in molecular diagnostic. Industrial biotechnology: applied to industrial and other production processes. Agricultural biotechnology: applied to agricultural processes and products. Marine biotechnology: marine and aquatic applications of biotechnology BIOTECHNOLOGY

3. Medical Biotechnology Medical Biotechnology is the use of living cells and cell materials to research and produce pharmaceutical and diagnostic products that help to treat and prevent human diseases. Applications Pharmacology: Insulin, GH, antibiotic, clotting factor, antibody production Gene therapy: Somatic/ germ line Stem cells Tissue engineering

4. Stem cell therapy: Bone marrow transplants, replacing damaged heart tissue after a heart attack and replacing damaged nerve tissue in spinal cord injury. A form of regenerative medicine, tissue engineering is the creation of human tissue outside the body for later replacement.

5. Recombinant DNA technology Manipulation of genetic material (DNA) in the laboratory involving isolation & end to end joining of DNA sequences from very different sources to make chimeric molecules are collectively referred to as rDNA technology. Other terms are Gene manipulation Genetic engineering Gene cloning Genetic modifications

6. Tools & techniques involved in rDNA technology Restriction enzymes (DNA cutting) DNA ligase (joining) Cloning (DNA amplification) Host cell (factories) Vector (vehicle) Gene transfer method (Transformation, Conjugation, direct transfer, electroporation) Library (collection of clones ) Probes Isolation & purification of NA Blotting & hybridization DNA sequencing PCR

7. Restriction enzyme Endonuclease Cut DNA at specific DNA sequences within the molecule Into unique, short pieces in a sequence specific manner (not randomly) Named after the bacterium from which they are isolated. [ EcoRI - from E. Coli] Each enzyme recognizes & cleaves a specific & double stranded DNA sequence 4-7 bp long Palindrome: same sequence from 5 ΄ 3 ΄ direction

8. Restriction site If recognize 4bp, cut once in every 256 bp (4 ⁴) If recognize 4bp, cut once in every 4096 bp (4 ⁶) The DNA cuts results in Blunt end Sticky end: useful in constructing hybrid/ chimeric DNA molecule Restriction enzyme and DNA ligase are used to prepare chimeric DNA molecules.

9. L ibrary A library - is a collection of recombinant clones Genomic library: prepared from total DNA of a cell line or tissue cDNA library: only expressed DNA [cDNA copies of mRNA] in a tissue

10. DNA cloning Production of a large number of identical copies of DNA of interest by introducing that foreign DNA into a replicating cell. Cloning vector DNA molecule to which the target DNA is joined Plasmid (present in all bacteria), Virus (phages) Artificial- cosmid , BAC, YAC Essential properties of vector Autonomously replicated within host cell Contain at least one specific restriction site Carry gene for selection (antibiotic resistance gene)

11. Steps of cloning Restriction enzymes (e.g. EcoRI ) cut the DNA to be amplified Bacterial plasmids (circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme producing complementary sticky end DNA ligase joins the DNA fragment of plasmid & DNA of interest to form a rDNA molecule The recombinant plasmid is introduced into host bacteria by transformation

12. 5. Bacteria are grown (in presence of antibiotic- so only transformed bacteria survive, the others die) 6. Bacteria are lysed, hybrid plasmid are isolated 7. Plasmids are cut with same restriction enzyme. Many copies of DNA of interest released

13. Probes Probe is a molecule used to detect the presence of a specific fragment of a DNA or RNA. Short piece (15-20 NT) of ssDNA or RNA, labeled with a radioisotope [ ³² P]or fluorescent dye Complementary to a sequence of the DNA of interest/ target DNA Used to screen a library for a complementary sequence in the coding region of the gene. If the sequence match exactly, probes will hybridize

14. Probes

15. Blotting & hybridization Blotting is a process of immobilization of sample nucleic acids on solid support (nitrocellulose membrane) The blotted nucleic acids are then used as targets in the hybridization experiments for specific detection Autoradiography allow visualization of specific DNA/RNA fragments Types- Southern blot (for DNA) Northern blot (for RNA) Western blot (for protein)

16. Blotting & hybridization

17. Southern blot : A method of transferring DNA from an agarose gel to nitrocellulose filter, on which the DNA can be detected by a suitable probe [cDNA or RNA ] Northern blot : A method of transferring RNA from an agarose gel to nitrocellulose filter, on which the RNA can be detected by a suitable probe [cDNA or RNA] Western blot : A method of transferring protein to a nitrocellulose filter, on which the protein can be detected by a suitable probe [ antobody ]

18. Blot transfer procedure

19. Determination of DNA sequences

20. Polymerase chain reaction PCR is a enzymatic, test tube method of amplifying a target sequence of DNA PCR can amplify Very small quantity of DNA (1 part in million) DNA from any source: bacterial, viral, plant Very rapidly (each cycle 5-10 min; 20 cycle made 10 ⁶ copies DNA from a single cell, hair follicle, spermatozoon

21. Steps of PCR Use DNA polymerase from Thermus Aquaticus [ TaqP ]. Heat stable, so not denatured at 70-80 ⁰C. Primer construction [20-35 NT] according to flanking sequence 1. Denaturation of DNA [94 ⁰C] 2. Annealing of primers to ssDNA [54 ⁰C] 3. Chain extension by DNA-P [72 ⁰C] New dsDNA molecules can be denatured & copied repeatedly

22. Steps of PCR

23. Autoradiograph

24. Uses of PCR Forensic analysis of DNA sample To detect infectious agent [latent virus-HIV] To make prenatal genetic diagnosis To detect allelic polymorphism To establish precise tissue types for transplants To study evolution, using DNA from archeological samples Quantitate RNA analysis [RT-PCR] To score in vivo protein DNA occupancy using chromatin immuno -precipitation assay to facilitate NGS [next generation occupancy] New uses are developed every year.

25. Polymorphism Change in genotype that does not affect phenotype or may change phenotype that is harmless. Primarily occur in non-coding sequence [only 2% genome encode protein in human] Genome of any 2 unrelated people are 99.5% identical Important tool in genome mapping localization of genes for genetic disorders determination the risk for disease paternity testing, criminal identification

26. Types of polymorphism SNPs – single nucleotide polymorphism Single base changes in DNA Most common – 1 in 350 bp RFLP- Restriction fragment length polymorphism STR- Short tandem repeats (Microsatellite/ simple) VNTRs- variable number tandem repeats

27. RFLP When genome variation affect restriction enzyme cleavage site. D etect human genetic variation VNTR VNTR is a location in a genome where a short nucleotide sequence is organized as tandem repeat

28. Applications of rDNA technology Helps in gene mapping, thus localizes specific gene on chromosome Understanding molecular basis of a number of disease. e.g.- Familial hypercholesterolemia, sickle cell disease, thalassaemia , cystic fibrosis, muscular dystrophy and also some complex multifactorial diseases (heart disease, cancer, DM) Commercial preparation of protein & hormone- insulin, growth hormone, TPA, blood clotting factor, interferon, interleukin

29. Production of human enzyme used in therapy of storage disease (e.g. Pompe’s , Gaucher’s disease) Preparation of several vaccines (Hepatitis B) DNA based diagnostic test can identify structural variants (point mutation), dysfunctional & absent genes (sickle cell disease, AIDS) This technology is used both to diagnose existing disease as well as to predict the risk of developing a specific disease and individual response to pharmacological therapeutics Special techniques have lead remarkable advances in forensic medicine

30. Make platform for sequencing of genomic DNA Gene therapy cure a number of inherited disorder & disease caused by somatic mutations (sickle cell disease, thalassemia, Duchene muscular dystrophy) Used in a number of analytic techniques- PCR, RFLP, DNA typing, Southern b, northern b, western blotting Trans genesis- of plant & animal to make more resistant to disease & infection Huge application in agricultural & livestock industries

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