Contenu connexe


Recombinant DNA.pptx

  1. Series of Lectures Course Title: Methods in Molecular Biology Course code: BCH-613 Credit Hours:3(1-2) Dr. Sumera Shaheen Assistant Professor Dept. of Biochemistry Govt.College Women University Faisalabad
  2. Theory: Introduction to recombinant DNA technology; Vectors, their types, cloning and expression vectors; Restriction and modifying enzyme, Polymerase chain reaction (PCR) - types; (inverse, touchdown, nested, hemi-nested, pit stop, multiplex, reverse transcriptase, RACE, real-time) and its applications; DNA sequencing technologies; Detection of mutations and/or SNPs; Analysis of nucleic acids by gel electrophoresis horizontal, vertical, pulse field, denaturing gradient gel electrophoresis; Expression of recombinant proteins and their purification by affinity chromatography; Generation of antibodies and their uses; enzyme-linked immunosorbent assay; Blotting: Southern, Western and Northern;
  3. Recommended Books 1. B. Lewin. 2017. GeneXII, 12th edition, Jones & Bartlett Learning 2. Robert F Weaver,2012 Molecular Biology. Mc GrawHills. 3. T. A. Brown. 2016. Gene Cloning and DNAAnalysis: An Introduction,7th Edition.. Wiley-Blackwell. 4. M. R Green and J. Sambrook. 2014. Molecular Cloning: A Laboratory Manual.4th Edition; Cold Spring Harbor Laboratory Press. 5. Recombinant DNA technology paper – July 19, 2013, by Keya Chaudary 6. Wilson K and Walker J, 2010. Principles and Techniques of Biochemistry and Molecular Biology. 7th Edition; Cambridge University Press. 7. Walker JM and Rapley, 2008. Molecular Biomethods Handbook (Methods in Molecular Biology). 2nd Edition; Humana Press. 9. S. B. Primrose and R. Twyman. 2006. Principles of Gene Manipulation and Genomics. 7th Edition; Wiley- SBlackwell. 10. F. M. Ausubel. 2005. Short Protocols in Molecular Biology (2 volume set). 5th Edition; John Wiley and Son.
  4. Lecture 1: Recombinant DNA Technology Dr.Sumera Shaheen Assistant Professor Dept. of Biochemistry
  5. Recombinant DNA Technology  It is a technology of “joining together of DNA molecules from two different species” (rDNA), that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry etc.  It involves procedures for analyzing or combining DNA fragments from one or several organisms including the introduction of the rDNA molecule into a cell for its replication, or integration into the genome of the target cell.
  6. cont  Focus of all genetics is the “gene”,  Fundamental goal of laboratory geneticists is to “isolate, characterize, and manipulate genes.”  Although it is relatively easy to isolate a sample of DNA (Consider the fact that each cell contains approximately 2 meters (6 feet) of DNA)  Therefore, a small tissue sample will contain many kilometers of DNA.  However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA  Enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.
  7. History of Genetic Engineering/Recombinant DNA technology  Paul Berg "father of genetic engineering“ Nobel Prize in 1980  In conjunction with his studies of the tumor virus SV40 (simian vacuolating virus 40), in 1972, he succeeded in inserting DNA from a bacterium into the virus' DNA.  He created the first DNA molecule made of parts from different organisms.  This type of molecule became known as "hybrid DNA" or "recombinant DNA".  Among other things, Paul Berg's method opened the way to creating bacteria that produce substances used in medicines.
  8. cont  In 1973, Herbert Boyer, University of California, and Stanley Cohen, at Stanford University  Constructed functional organisms that combined and replicated genetic information from different species.  They demonstrated the potential impact of DNA recombinant engineering on medicine pharmacology, industry and agriculture.  Boyer and Cohen's achievement represented an advance upon the ingenious techniques developed by Paul Berg, in 1972, for inserting viral DNA into bacterial DNA.
  9. cont  It was a creative synthesis of earlier research that made use of:  Living organisms able to serve as carriers for genes from another organism.  Enzymes to cleave and rejoin DNA fragments that contain such genes.  DNA molecules from one organism precisely targeted and manipulated for insertion into the DNA of another organism
  10. Scientific events lead to development of rDNA technology Event Year Mendel’s experiments published 1866 DNA discovered in cell 1869 Mutation of genes by x-rays 1927 One gene-one enzyme hypothesis 1941 DNA is identified as the genetic material 1944 Structure of DNA determined 1953 Ribosomes synthesize protein 1954 Function of mRNA proposed 1961 Genetic code determined 1961-64 Isolation of a restriction enzyme 1970 Recombinant DNA techniques developed Early 1970s Isolation of a single copy gene from higher eukaryote 1977 Rapid method of DNA sequencing developed 1977 Plant transformation 1983 Field testing of transformed plants ca. 1986 Release of engineered plants to general public in the US 1995-96
  11. Basic Principle of Recombinant DNA Technology  The DNA is inserted into another DNA molecule called vector.  The recombinant vector is then introduced into a host cell where it replicates itself and multiple copy of gene are produced
  12. Steps of Recombinant DNA Technology 1. Isolation and purification of Gene of interest (DNA fragment) 2. Cutting of DNA fragment and Vector with Restriction Enzymes (endonucleases) 3. Desired gene is inserted into a vector (plasmid, bacteriophage or a viral genome) 4. Plasmid transferred to host cell (bacteria, yeast, plant or animal cell) 5. After entering the host cell, vector grown/replicates as host cell replicates, recombinant molecules are passed on to progeny known as ‘clones’ 6. Large quantities of Cloned DNA can be recovered & analyzed from the host cells after processing
  13. Tools Used in Recombinant DNA Technology 1. Purified Gene 2 Restriction Enzymes 3. Ligase (Enzymes which join together two DNA molecules) 4. Vectors (carrier` of DNA molecule) 5. Suitable host (bacteria, yeast etc.)
  14. 1-Purified Gene  Isolation of DNA  Amplification of the desired gene  Purification of desired gene
  15. 2-Restriction Enzymes  Naturally produced by bacteria – Restriction Endonucleases (RE):  A class of endonucleases that cleaves/cut DNA only within or near those sites which have specific base sequences, are known as restriction enzymes  Natural function in bacteria - destroy bacteriophage DNA which attack bacterial cells  Cannot digest host DNA with methylated C (cytosine)
  16. Site recognised by them are called recognition sequences or recognition sites. There are three types of RE. Type I Restriction Endonucleases Type II Restriction Endonucleases Type III Restriction Endonucleases Type II are used because they cut the sequence which they recognize
  17. Some Restriction Enzymes
  18. 3-Ligase Enzyme DNA ligase is a enzyme that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA).  Naturally DNA ligase has applications in both DNA replication and DNA repair  Needs ATP  DNA ligase has extensive use in molecular biology laboratories for genetic recombination experiments
  19. 4-Vectors “A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cells and serve as a vehicle that carry DNA fragment or insert to be cloned.” Therefore, a vector must have an origin of DNA replication (ori) that functions in the host cell.
  20. Types of Vectors Used Plasmids (5-10 kb)  pUC18/19 (2686 bp)  In host it replicates 500 copies per cell and contains several RE sites Bacteriophage (10-15 kb)  Lambda (able to replace 1/3 of the chromosome with large pieces of foreign DNA)  M13 Cosmids (50 kb)  Hybrid vectors, part plasmid – part lambda BACs & YACs (300 kb, up to 1,000 kb)  Artificial chromosome
  21. 5-Host for Recombinant DNA Technology 1. Bacteria - E. coli - used because is easily grown and its genomics are well understood. Gene product is purified from host cells 2. Yeasts - Saccharomyces cerevisiae Used because it is easily grown and its genomics are known. May express eukaryotic genes easily. Gene product easily collected and purified 3. Plant cells and whole plants- May express eukaryotic genes easily. Plants are easily grown . 4. Mammalian cells May express eukaryotic genes easily. Difficult to work with.
  22. Applications of Recombinant DNA Technology 1. Scientific applications  Many copies of DNA can be produced  Increase understanding of DNA  Identify mutations in DNA  Alter the phenotype of an organism 2. Diagnose genetic disease
  23. Therapeutic Applications  Produce human proteins – hormones and enzymes (Insulin, hGH, INFα, INFβ and INFγ)  Gene therapy, antiviral therapy, vaccination, synthesizing clotting factors, drug delivery to target sites  Vaccines : Cells and viruses can be modified to produce a pathogen’s surface protein (Influenza; Hepatitis B; Cervical cancer vaccine)
  24. Agricultural Applications Growing crops of your choice (GM food). Cells from plants with desirable characteristics can be cloned to produce many identical cells, then can be used to produce whole plants from which seeds can be harvested. Pesticide resistant crops, Fruits with attractive colors, all being grown in artificial conditions.
  25. Genetic fingerprinting identification  DNA fingerprinting to identify the source of bacterial or viral pathogens  Bioterrorism attacks (Anthrax in U.S. Mail)  Medical negligence (Tracing HIV to a physician who injected it)  Outbreaks of foodborne diseases Nanotechnology  Bacteria can make molecule-sized particles  Bacillus cells growing on selenium form chains of elemental selenium
  26. Other uses: fluorescent fishes, glowing plants etc.
  27. Advantages of Recombinant technology:  Provide substantial quantity of the desired product  No need for natural or organic factors  Tailor made product that you can control  Unlimited utilizations  Cheap  Resistant to natural inhibitors
  28. Disadvantages of Recombinant technology:  Commercialized and became big source of income for businessmen  Effects natural immune system of the body  Can destroy natural ecosystem that relies on organic cycle. Prone to cause mutation that could have harmful effects  Major international concern: manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype  Concerns of creating super‐human race