Methods in Biotechnology in plant breeding.pptx

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1. Techniques in Molecular Bi ology Dr. Sammyia Jannat Department of Biotechnology University of Kotli Azad Jammu and Kashmir 1

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3. Course Content Introduction to recombinant DNA technology; restriction and modifying enzymes; cloning and expression vectors and their types; Transformation Methods expression of recombinant proteins and their purification by affinity chromatography; polymerase chain reaction (PCR) - types; (inverse, touch-down, nested, hemi-nested, pit stop, multiplex, reverse transcriptase, RACE, real-time) and its applications; detection of mutations and/or SNPs; DNA fingerprinting; analysis of nucleic acids by gel electrophoresis – horizontal, vertical, pulse field, denaturing gradient gel electrophoresis; analysis of proteins by native and SDS-PAGE; 2-D gels; generation of antibodies and their uses; enzyme-linked immunosorbant assay; Southern , Western, Northern blotting; DNA sequencing 3

4. Practical Preparation of stock and working solutions; isolation of nucleic acids and their quantification; restriction digestion of DNA and preparation of restriction maps; gel electrophoresis; polymerase chain reaction (PCR); detection of mutations by restriction fragment length polymorphism; preparation of chemically competent cells; transformation of bacteria with plasmid DNA; analysis of proteins by SDS-PAGE 16S rDNA sequence analysis through Basic local alignment search tool (BLAST ); 16S rDNA sequence submission guidelines in NCBI GenBank database. 4

5. Recommended Books 1 . Ausubel FM, 2005. Short Protocols in Molecular Biology (2 volume set).5 th Edition; John Wiley and Son. 2. Green MR and Sambrook J, 2001. Molecular Cloning: A Laboratory Manual. 3 rd Edition; Cold Spring Harbor Laboratory Press. 3. Primrose SB and Twyman R, 2006. Principles of Gene Manipulation and Genomics.7 th Edition; Wiley-Blackwell. 4. Wilson K and Walker J, 2010. Principles and Techniques of Biochemistry and Molecular Biology.7 th Edition; Cambridge University Press. 5. Walker JM and Rapley , 2008. Molecular Biomethods Handbook (Methods in Molecular Biology). 2 nd Edition; Humana Press. 5

6. Recombinant DNA Technology the artificial manipulation, modification, and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms. The term genetic engineering is generally used to refer to methods of recombinant DNA technology, which emerged from basic research in microbial genetics. The techniques employed in genetic engineering have led to the production of important products, including human insulin, human growth hormone, and hepatitis B vaccine, as well as to the development of genetically modified organisms such as disease-resistant plants. 6

7. The term genetic engineering initially referred to various techniques used for the modification or manipulation of organisms through the processes of heredity and reproduction. As such, the term embraced both artificial selection and all the interventions of biomedical techniques: A rtificial insemination, I n vitro fertilization (e.g., “test-tube” babies), Cloning, G ene manipulation. 7 G enetic Engineering

8. In the later part of the 20th century, however, the term came to refer more specifically to methods of recombinant DNA technology (or gene cloning), in which DNA molecules from two or more sources are combined either within cells or in vitro and are then inserted into host organisms in which they are able to propagate 8

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10. History The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome . An organism that is generated through genetic engineering is considered to be genetically modified (GM) and the resulting entity is a genetically modified organism (GMO) 10

11. Restriction enzymes : Discovered in the 1960s–1970s , key figures were Werner Arber, Hamilton Smith, and Daniel Nathans . DNA ligase : Discovered in 1967 independently by Robert Lehman and Martin Gellert . Together , these two discoveries enabled recombinant DNA technology (1973, Cohen & Boyer) , which is the foundation of genetic engineering. 11

12. In 1972, Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus. In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism (bacteria) by inserting antibiotic resistance genes into the plasmid of an Escherichia coli bacterium. A year later Rudolf Jaenisch , in 1974, created a transgenic mouse by introducing foreign DNA into its embryo, making it the world's first transgenic animal. The first company to focus on genetic engineering, Genentech, was founded in 1976 and started the production of human proteins. Genetically engineered human insulin was produced in 1978 and insulin-producing bacteria were commercialized in 1982. These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe . 12

13. Genetically modified food has been sold since 1994, with the release of the Flavr Savr tomato. The Flavr Savr was engineered to have a longer shelf life, but most current GM crops are modified to increase resistance to insects and herbicides . GloFish , the first GMO designed as a pet, was sold in the United States in December 2003. In 2016 salmon modified with a growth hormone were sold 13

14. In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and Robert Swanson A year later the company produced a human protein ( somatostatin ) in E.coli . Genentech announced the production of genetically engineered human insulin in 1978 . In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented. The insulin produced by bacteria was approved for release by the Food and Drug Administration (FDA) in 1982 . 14

15. 15 Glofish Flavr - Savr Tomato

16. In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorisation to perform field tests with the ice-minus strain of Pseudomonas syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges. In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment when a strawberry field and a potato field in California were sprayed with it . Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher ". 16

17. The first field trials of genetically engineered plants occurred in France and the US in 1986, tobacco plants were engineered to be resistant to herbicides. The People's Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992. In 1994 Calgene attained approval to commercially release the first genetically modified food, the Flavr Savr , a tomato engineered to have a longer shelf life. In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil , making it the first genetically engineered crop commercialized in Europe. In 1995, Bt Potato was approved safe by the Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the US. In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the US, Brazil, Argentina, India, Canada, China, Paraguay and South Africa . 17

18. In 2010, scientists at the J. Craig Venter Institute created the first synthetic genome and inserted it into an empty bacterial cell. The resulting bacterium, named Mycoplasma laboratorium , could replicate and produce proteins. Four years later this was taken a step further when a bacterium was developed that replicated a plasmid containing a unique base pair, creating the first organism engineered to use an expanded genetic alphabet. In 2012, Jennifer Doudna and Emmanuelle Charpentier collaborated to develop the CRISPR/Cas9 system, a technique which can be used to easily and specifically alter the genome of almost any organism . 18

19. Genetic engineering has been applied in numerous fields including research, medicine, industrial biotechnology and agriculture. In research GMOs are used to study gene function and expression through loss of function, gain of function, tracking and expression experiments. By knocking out genes responsible for certain conditions it is possible to create animal model organisms of human diseases. As well as producing hormones, vaccines and other drugs, genetic engineering has the potential to cure genetic diseases through gene therapy. The same techniques that are used to produce drugs can also have industrial applications such as producing enzymes for laundry detergent, cheeses and other products. 19

20. Two different school of thoughts The rise of commercialized genetically modified crops has provided economic benefit to farmers in many different countries, but has also been the source of most of the controversy surrounding the technology. This has been present since its early use; the first field trials were destroyed by anti-GM activists. Although there is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, GM food safety is a leading concern with critics. Gene flow, impact on non-target organisms, control of the food supply and intellectual property rights have also been raised as potential issues. These concerns have led to the development of a regulatory framework, which started in 1975. It has led to an international treaty, the Cartagena Protocol on Biosafety, that was adopted in 2000. Individual countries have developed their own regulatory systems regarding GMOs, with the most marked differences occurring between the US and Europe. 20