Genetic engineering principle, tools, techniques, types and application

Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.

Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR

1. GENETIC ENGINEERING Principle, Tools, Techniques, Types and Application Presented by: Mr. TARUN KAPOOR Assistant Professor, Shri Ram College of Pharmacy, Karnal

2. Contents Genetic Engineering : An Introduction & Basic principles Tools of Genetic Engineering (Enzymes and transformation factors) Cloning vectors , restriction endonucleases and DNA ligase Methods of Gene transfer Methods of DNA amplification (Gene Cloning and PCR) Recombinant DNA technology & its Application Recombinant Pharmaceuticals : An Overview Production of genetic engineering products ( Interferon, Vaccines- hepatitis- B, Insulin ) Brief introduction to PCR : Principle, Advantages and Applications

3. Genetic Engineering Genetic Engineering involves manipulation of genetic material, is also called Recombinant DNA technology or Gene Cloning. Genetic recombination technology consists of the breakage and joining of DNA molecules . Genetic engineering primarily involves the manipulation of genetic material ( DNA) to achieve the desire goal in pre determined way. Other terms are Recombinant DNA technology, Gene manipulation, Gene cloning, Genetic modifications.

4. Basic principle of Genetic Engineering DNA fragment of interest is obtained by cleaving chromosomes by Restriction endonuclease. Cloning vector is cleaved with Restriction endonuclease. Fragments are ligated to the prepared cloning vector. Recombinant vector DNA is introduced into the host cell Propagation ( cloning) produces many copies of recombinant DNA The gene is extracted and harvested the product

5. Tools of Genetic Engineering Enzymes Restriction Endonucleases (REs ) DNA ligase DNA Polymerases Reverse transcriptase Transformation factors Plasmid Bacteriophage Cosmid Yeast

6. Restriction Endonucleases (REs ) Used as mol. scissors to cut DNA –DNA at specific DNA sequences To generate a set of smaller fragments Recognize specific DNA sequences called “ palindrome” ( restriction sites ) Example : EcoR I recognizes sequence cuts the Phosphodiesterase bonds of the DNA on both the strands

7. Plasmid Plasmid with a cut RE DNA RE 9 DNA fragment

8. Applications of RE Sequencing of DNA Cloning of DNA Antenatal diagnosis of inherited disorders DNA finger printing (having forensic applications) For Southern blot technique (for detecting the presence of a particular base sequence in the sample DNA )

9. DNA ligase Joins two DNA molecules or fragments . DNA Polymerases Synthesis of DNA using DNA template Reverse transcriptase Enzyme found in retroviruses that makes DNA copy, using RNA as template

10. Transformation factors Into the DNA of the vector a foreign DNA can be inserted, integrated/incorporated . Use : For amplification by cloning and for gene therapy. Plasmid, Bac t eriopha g e , Cosmid, Yeast

11. C lo n i n g Ve c to rs Vectors are the DNA molecule, which can carry A foreign DNA fragment to be cloned. The are self replicating in an appropriate host cell. The most important vectors are Plasmids, Bacteriophages & Cosmid. An ideal characteristics of an vector is should be small in size with endonuclease site .

12. Plasmid A small, circular, dsDNA present in bacteria Confer antibiotics resistance against the bacteria Many copies of plasmid in a bacterium Replicate independent of the bacterial DNA .

13. Cosmid: Cosmid are vectors posses the characteristic of both plasmid and bacteriophage. Can carry larger DNA fragments Cosmid can be constructed by adding a fragment of DNA to plasmid .

14. Yeast Artificial Chromosomes (YAC) is a synthetic DNA that can accept large fragment (particular human DNA ). It is possible to clone large DNA pieces by using YAC . Bacteriophage: It is a virus that can infect bacteria .

15. Methods of gene Transfer Transformation Transduction Electroporation Conjugation Microinjection Liposome mediated gene transfer

16. Transformation

17. Electroporation: Application of high voltage electrical field to cells Direct transfer: Micro injection Particle bombardement Liposome mediated gene transfer

18. Types of DNA amplification Cloning In vivo method using bacteria Used to amplify longer segments of DNA Suitable for large scale protein production Polymerase Chain Reaction (PCR ) In vitro method using DNA polymerase Shorter segments of DNA can be amplified Shorter time for amplifying DNA fragments

19. DNA Amplification Production of many identical copies of a DNA fragment of interest. Uses Further DNA analysis For large-scale genetic expression Protein production

20. Cloning Production of an identical copy of either DNA or a cell or an organism is called cloning . It is of 2 Types: Molecular cloning : Production of identical DNA molecules (i.e ., identical in base-sequence) Somatic cloning : Production of cells or organisms with identical genetic makeup

21. Genetic recombination is exchange of information between two DNA segments within same species. But artificially when a gene of one species is transferred to another living organism, it is called recombinant DNA technology or genetic engineering . rDNA: Production of a unique DNA molecule by joining together two or more DNA fragments which are derived from different biological sources. rDNA technology : A series of procedures used to recombine DNA segments. Under certain conditions, a recombinant DNA molecule can enter into cell & replicate.

22. History of r DNA technology Recombinant DNA technology is one of the recent advances in biotechnology , which was developed by two scientists named Boyer and Cohen in 1973.

23. Applications of rDNA Technology 1. L a r g e s ca le p r o d u cti o n o f h u m a n p r o t e i ns by genetically engineered bacteria. Recombinant human insulin Recombinant human growth hormone Recombinant blood clotting factors Recombinant hepatitis B vaccine Cytokines and growth factors (IF, IL) Monoclonal antibodies Recombinant enzymes Recombinant HIV protein for ELISA testing Albumin , fibrinolytic and thrombolytic agents

24. Basic research – understanding structure and functions of DNA & proteins ( Human Genome Project ) Gene therapy for genetic diseases Food production Plant: Genetically modified corn Forensic applications Genetically modified organisms are called transgenic organisms. Mice (Study human immunity), Chicken (Resistant to infection), Cows (Increase milk & leaner meat) Applications in ecology: Recombinant Bacteria which can be engineered to “ eat” oil spills .

25. Recombinant Pharmaceuticals Human Insulin Human Growth Hormone Human blood clotting factors Vaccines Monoclonal Antibodies Interferons Antibiotics & other secondary metabolites

26. Human Insulin Insulin is a hormone produced by β-islets of Langerhans of pancreas. It was discovered by sir Edward Sharpey Schafer (1916) while studying Islets of Langerhans. People who do not produce the necessary amount of insulin have diabetes . Chemically, insulin is protein consist of 51 amino acids, 30 construct polypeptide chain B and 21 amino acids construct polypeptide chain A and both chains linked by disulfide bond.

27. Modify E.coli cells to produce insulin; performed by Genentech in 1978 Prior , bovine and porcine insulin used but induced immunogenic reactions. Also , there were many purification and contamination hassles. To overcome these problems, researchers inserted human insulin genes into a suitable vector (E.coli ). First , scientists synthesized genes for the two insulin A & B chains. Then inserted into plasmids . The genes were inserted in such a way that the insulin & B- galactosidase residues would be separated by a methionine residue. This is so that the insulin A & B chains can be separated easily by adding cyanogen bromide .

28. Producing Recombinant Insulin The vector was then transformed into E.coli cells . Once inside the bacteria, the genes were "switched-on" by the bacteria to translate the code into either the "A" chain or the "B" chain proteins found in insulin The purified insulin A and B chains were then attached to each other by disulphide bond formation under laboratory conditions

29. Hepatitis B Vaccine Hepatitis B virus (HBV) is common infectious diseases. WHO estimates that there are 285 million chronic carriers of HBV worldwide. Hepatitis B is 50 to 100 times more infectious than AIDS. Hepatitis B is irritation and swelling ( inflammation) of liver due to infection with hepatitis B virus (HBV). Other types include : Hepatitis A , C and D. It produces several chronic liver disorders such as Liver cirrhosis and primary liver cancer. Hepatitis B Recombinant Vaccine : It’s a novel and significant developed vaccine which is produced from the antigenic proteins of Hepatitis B virus by recombinant process that duplicates the chemical messages and secreted factors (Interleukin-2) for the communication and activity of immune cells .

30. Production of recombinant HBV Vaccine Production of these genes is needed in order to get production of vaccines on a large scale. A general procedure for the production of recombinant Hepatitis B vaccines are described here- HBs antigen producing gene is isolated from HB virus by isolation process (cell lysis , protein denaturation, precipitation , centrifugation and drying). A plasmid DNA is extracted from a bacterium- E.coli and is cut with restriction enzyme- Eco RI forming the plasmid vector. The isolated HBs antigen producing gene is located and inserted into the bacterial plasmid vector on forming the recombinant DNA.

31. This recombinant DNA, containing the target gene, is introduced into a yeast cell forming the recombinant yeast cell. The recombinant yeast cell multiplies in the fermentation tank and produces the HBs antigens. After 48 hours, yeast cells are ruptured to free HBsAg . The mixture is processed for extraction. The HBs antigens are purified. HBsAg are combined with preserving agent and other ingredients and bottled. Now it is ready for vaccination in humans .

32. INTERFERONS Interferons (IFNs) were the first family of cytokines to be discovered. In 1957 researchers observed that if susceptible animal cells were exposed to a colonizing virus, these cells immediately become resistant to attack by other viruses. This resistance was induced by a substance secreted by virally-infected cells , which was named ‘interferon’ (IFN ). Humans produce at least three distinct classes, IFN-a, IFN-b and IFN-g. Biological effects : Induction of cellular resistance to viral attack. Regulation of most aspects of immune function. Regulation of growth and differentiation of many cell types. Sustenance of early phases of pregnancy in some animal species.

33. Production of Interferons A DNA sequence coding for the product was synthesized and inserted into E. coli. The recombinant product accumulates intracellularly as inclusion bodies. Large-scale manufacture entails an initial fermentation step. After harvest, the E. coli cells are homogenized and the inclusion bodies recovered via centrifugation. After solubilization and refolding, the interferon is purified to homogeneity by a combination of chromatographic steps. The final product is formulated in the presence of a phosphate buffer a nd sodium chloride. It is p resented as a 30 mg/ml solution in glass vials and displays a shelf- life of 24 months when stored at 2–8°C.

34. Human Fibroblast Human Interferon β Gene Modified Human Interferon β Gene Plasmid Restriction enzyme cut Plasmid E. Coli containing its Own gene E. Coli containing rDNA Replicated E. Coli producing IFN β Purifie d Interferon 1 β Packed and Ready for Use rDNA

35. Polymerase Chain Reaction PCR is an in vitro technique for the amplification of DNA . Developed by Kary Mullis in the 1980s Much faster More sensitive method than cloning. Very little DNA sample is sufficient Can only amplify short segments of DNA Cannot be used for amplifying genes and for production of proteins

36. Principle Double stranded DNA of interest is denatured to separate strands Each strand is then allowed to hybridize with a primer. The primer template duplex is used for synthesis . Program thermocycler for times include three steps : Denaturation, anneling and extension repeated again and again to generate multiple forms of target DNA. The primer extension product synthesized in 1 cycle serve as template for next cycle.

37. Procedure A mixture of DNA sample + dNTP + Primer + Enzyme : Taq DNA polymerase Treatment of the mixture :1 cycle 94 – 95˚ C : Denaturation of DNA : 30 – 60 sec 52 – 54˚ C : Annealing of primers : 30 – 60 sec 72˚ C : Extension of the DNA : 1 min

38. Types of PCR: Real-time PCR Nested PCR Inverse PCR Reverse transcription PCR Advantages of PCR Very little DNA sample is required Amplification time is very short . Amplification rate is high. Uses: W hen insufficient DNA molecules are present in test samples for DNA analytical techniques.

39. Applications of PCR Diagnosis : Bacterial and viral diseases ( TB , Hepatitis C , HIV) Medicolegal / Forensic cases: DNA amplification from hair, saliva, semen and blood sample ( DNA fingerprinting ) Diagnosis of genetic disorders: SCD, thalassemia, cystic fibrosis Prenatal diagnosis of inherited disorders Cancer detection: To monitor abnormal cells present in treated patients. Identification of mutation in oncosuppressor genes Fossil studies: T o study evolution by comparing the sequences in the extinct and living organism