LECTURE 4 Recombinant DNA Technology lec# 1.pptx

Reccombenant DNA technology

1. RECOMBINANT DNA TECHNOLOGY LEC 1 SYED MUHAMMAD HASSAN ASKRI

2. A series of procedures used to recombine DNA segments . Under certain conditions, a recombinant DNA molecule can enter a cell and replicate .

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

4. RECOMBINANT DNA   RECOMBINANT DNA:-   DNA molecules constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell

5. The DNA is inserted into another DNA molecule called ‘vector’ The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced

6. Basic principle of recombinant DNA technology

7. How is Recombinant DNA made? There are three different methods by which Recombinant DNA is made. They are Transformation, Phage Introduction, and Transformation. Non-Bacterial

8. Transformation The first step in transformation is to select a piece of DNA to be inserted into a vector. The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules. The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. coli . The host cells must be specially prepared to take up the foreign DNA.

9. Non-Bacterial transformation Microinjection, the DNA is injected directly into the nucleus of the cell being transformed. The host cells are bombarded with high velocity micro-projectiles, such as particles of gold or tungsten that have been coated with DNA.

10. Phage Introduction Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packaging of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.

11. How does rDNA work? Recombinant DNA works when the host cell expresses protein from the recombinant genes. A significant amount of recombinant protein will not be produced by the host unless expression factors are added. Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the transcription and translation of the gene by the cell. These signals include the promoter, the ribosome binding site, and the terminator.

12. Expression vectors, in which the foreign DNA is inserted, contain these signals. Signals are species specific. In the case of E. coli , these signals must be E. coli signals as E. coli is unlikely to understand the signals of human promoters and terminators. Problems are encountered if the gene contains introns or contains signals which act as terminators to a bacterial host. This results in premature termination, and the recombinant protein may not be processed correctly, be folded correctly, or may even be degraded.

13. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi. The uses of animal cells is difficult due to the fact that many need a solid support surface, unlike bacteria, and have complex growth needs. However, some proteins are too complex to be produced in bacterium, so eukaryotic cells must be used.

14. Large-scale production of human proteins by genetically engineered bacteria. Such as : insulin, Growth hormone, Interferons and Blood clotting factors (VIII & IX)

15. 1) Obtaining the human insulin gene Human insulin gene can be obtained by making a complementary DNA (cDNA) copy of the messenger RNA (mRNA) for human insulin.

16. 2)Joining the human insulin gene into a plasmid vector The bacterial plasmids and the cDNA are mixed together. The human insulin gene (cDNA) is inserted into the plasmid through complementary base pairing at sticky ends.

17. 3)Introducing the recombinant DNA plasmids into bacteria The bacteria E.coli is used as the host cell. If E. coli and the recombinant plasmids are mixed together in a test-tube.

18. 4)Selecting the bacteria which have taken up the correct piece of DNA The bacteria are spread onto nutrient agar . The agar also contains substances such as an antibiotic which allows growth of only the transformed bacteria.

19. Vaccine development The surface antigen of falciparum , one of the 4 species of malaria, has been transferred to E. coli to produce amounts large enough to develop a vaccine against this form of malaria. It works well enough for people who will visit a malarious region for a relatively short period of time. Plasmodium

20. Hemophilia A and B The genes encoding factors 8 and 9 are on the X chromosome. Like other X-linked disorders, hemophilia A and B are found almost exclusively in males because they inherit just a single X chromosome, and if the gene for factor 8 (or 9) on it is defective, they will suffer from the disease. There are many different mutant versions of the genes for factors 8 and 9 . Although some produce only a minor effect on the function of their protein, others fail to produce any functioning clotting factor.

21. Transferring the gene for normal adult hemoglobin into marrow stem cells of an individual with sickle-cell anemia. The goal is to promote the growth of enough cells to produce enough normal hemoglobin to alleviate the symptoms of sickle-cell anemia. Gene therapy for genetic diseases

22. Safety Issues in relation to Recombinant DNA Technology As bacteria is commonly used in recombinant DNA work, there has always been a concern among scientists and a worry among people that there is a possibility that a clone of highly pathogenic recombinant bacteria were made by accident, then escaped from the laboratory and caused an epidemic for which no drugs were available. Recombinant DNA Advisory Committee (RAC) was established in 1974 in the United States, which responds to public concerns regarding the safety of manipulation of genetic material through the use of recombinant DNA techniques.

23. 2 types of control : physical containment containment and biological Effective biological safety programs were operated in a variety of laboratories, which include a set of standard practices generally used in microbiological laboratories, and special procedures, equipment and laboratory installations that provide physical barriers of varying degrees.

24. In considering biological containment, the vector (plasmid, organelle, or virus) for the recombinant DNA and the host (bacterial, plant, or animal cell) in which the vector is propagated in the laboratory will be considered together . (i) survival of the vector in its host outside the laboratory, and (ii) transmission of the vector from the propagation host to other non- laboratory hosts. Biological containment

25. It is always possible that an antibiotic-resistant plasmid could be accidentally incorporated into a dangerous pathogen with serious medical consequences.

26.   Within living cells, the exchange of DNA sequences and genetic information can occur through a regulated series enzymatic reactions involving pairing of DNA molecules and phosphodiester bond breakage and rejoining. This type of sequence rearrangement is known as genetic recombination. genetic recombination responsible for rearranging sequences between different pieces of DNA, shaping the genome by altering the sequences that are present, pairing chromosome before cell division and promoting DNA repair.

27. DNA Recombination   Roles   Types

28. Biological Roles for Recombination 1. Generating new gene/allele combinations (crossing over during meiosis) 2. Generating new genes (e.g., Immuno - globulin rearrangement) Integration of a specific 3. 4. DNA element DNA repair

29. Practical Uses of Recombination 1. Used to map genes on chromosomes (recombination frequency proportional to distance between genes) 2. Making transgenic cells and organisms

30. THANKS