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Biotechnology

Biotechnology. Introduction. Biotechnology is essentially the use of living organisms and their products for health, social or economic purposes.

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Biotechnology

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  1. Biotechnology

  2. Introduction • Biotechnology is essentially • the use of living organisms and their productsfor health, social or economic purposes. • Biotechnology is widely considered to be the growth technology of the 21st centurywhich will lead to huge growth in the Biotechnology industry and exciting opportunities for graduates

  3. Application of Biotechnology • Its use and application ranges from fields like agriculture to industry (food, pharmaceutical, chemical, bioproducts, textiles etc.), medicine, nutrition, environmental conservation, animal sciences etc. making it one of the fastest growing fields. • The work is generally carried out in the laboratories, as it is a scientific research oriented field.

  4. Application of Biotechnology Applications of biotechnology are widespread, including the following: • diagnosis and treatment of human diseases. • improved production of therapeutic agents. • development of improved crop plant species. • Development of improved farm animals • development of improved pest/pathogen control processes

  5. Application of Biotechnology • development of biosensors for environmental pollutants. • development of improved waste treatment processes and methods for remediation contaminated sites. • production of transgenic organisms for production of new drugs, improved transplantation success and improved animal and plant.

  6. Selective Breeding • Humans use selective breeding, which takes advantage of naturally occurring genetic variation in plants, animals, and other organisms, to pass desired traits on to the next generation of organisms

  7. Selective Breeding • Breed only those plants or animals with desirable traits • People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.

  8. Selective Breeding • Nearly all domestic animals -- including horses, cats, and farm animals – and most crop plants have been produced by selective breeding No freaking way!

  9. Hybridization • Louis Burbank was the greatest selective breeder of all time. He developed the disease-resistant potato and more than 800 varieties of plants.

  10. Louis Burbank used the technique of hybridization and bred dissimilar individuals to combine the best traits of both parents. • The hybrids produced by these crosses were hardier than their parents

  11. Inbreeding • To maintain the desired characteristics of a line of organisms, breeders often use the technique of inbreeding. • Inbreeding is the continued breeding of individuals with similar characteristics

  12. Increasing Variation • In order for selective breeding to be successful, there must be a lot of genetic variation in the population • Breeders increase the genetic variation in a population by inducing mutations, which are the ultimate source of genetic variability

  13. Increasing Variation • Breeders increase the mutation rate by using radiation and chemicals

  14. Molecular Biology

  15. Molecular Biology • Molecular biology refers to the field of study regarding the investigation and manipulation of biological structures, processes, and phenomena at the molecular level. • Involves several classical basic techniques such as restriction enzymes, gel electrophoresis, and PCR, as well as more complex methods such as DNA fingerprinting, DNA sequencing, and genetic engineering

  16. Outline • Restriction-enzyme analysis • Gel electrophoresis • The polymerase chain reaction (PCR) • DNA Fingerprinting • DNA sequencing • Blotting techniques • Recombinant DNA

  17. Restriction Enzyme Analysis • aka restriction endonucleases • Recognizes specific base sequences and cleave the nucleic acid • PALINDROMES • Two-fold rotational symmetry • Generates fragments of DNA

  18. Restriction Enzyme and Gel Electrophoresis • DNA fragments produced by restriction enzymes can be separated by gel electrophoresis • Agarose (>20 kb) • PAGE (1 kb) • Visualization • Autoradiography • Ethidium bromide

  19. Gel Electrophoresis • A technique for separating DNA (or protein) molecules on the basis of size. DNA plus restriction enzyme Power source Longer fragments Shorter fragments Gel Mixture of DNA fragments

  20. Gel Electrophoresis • Utilizes agarose or polyacrylamide gels for the separation of DNA DNA plus restriction enzyme Power source Longer fragments Shorter fragments Gel Mixture of DNA fragments

  21. Electrophoresis

  22. Electrophoresis • Electrophoresis is used to map the structure of a DNA fragment

  23. Electrophoresis • Stained gel result • Visualization may be achieved through UV dyes or radioactive agents

  24. Polymerase Chain Reaction • A rapid and versatile in vitro method to amplify defined target DNA within a heterogeneous collection of DNA sequences (genomic DNA or cDNA)

  25. PCR Requirements • Template (genomic DNA or cDNA population) • Oligonucleotide primers • DNA polymerase (Taqpolymerase) • dNTP • Thermal cycler

  26. Cycles (25 – 30) • Denaturation • 95o C • Separate strands • DNA Synthesis • 70 – 75 oC (ideal temp for Taqpolymerase) • Thermusaquaticus (Taq) • Annealing • 50 – 70 oC (~5o C lower than Tm)

  27. Utility of PCR in Medical Diagnostics • Detection of bacteria and viruses by specific primers • HIV virus in people who have not mounted an immune response • Mycobacterium tuberculosis bacilli • Detection of certain cancer cells • ras genes and leukemias caused by chromosomal rearrangement • Monitoring cancer chemotherapy

  28. Utility of PCR in Forensics • DNA fingerprinting • Restriction fragment length polymorphisms • PCR-Based analysis • Can be used to determine biological parentage • Can be used to settle assault and rape cases

  29. DNA Fingerprinting: A tool for forensics and paternity cases • DNA analysis can be used for catching criminals, establishing parentage, finding how closely organisms are related and many other applications. • The pattern of bands in a gel electrophoresis is known as a DNA fingerprint or a‘genetic fingerprint’ or ‘genetic profile’ • If a DNA fingerprint found in a sample of blood or other tissue at the scene of a crime matches the genetic fingerprint of a suspect, this can be used as evidence • A DNA sample can be obtained from the suspect using blood, cheek epithelial cells taken from the mouth lining or even the cells clinging to the root of a hair

  30. Steps: • Get DNA sample • Amplify with PCR • Cut with restriction enzyme • Run resulting fragments on gel electrophoresis • Analyze result • A sample with the shorter DNA fragments travels through the gel faster than a sample with the larger fragments

  31. DNA profiles V S S1 S2 S3 V Victim S Sample from crime scene S1 Suspect 1 S2 Suspect 2 S3 Suspect 3 More than 20 fragments from Suspect 1 match those taken from the crime scene

  32. Genetic fingerprint of … 1 mother 2 child 3 possible father A 4 possible father B There is a match between one of the child’s restriction fragments and one of the mother’s. There is also a match between the child’s other fragment and one from possible father A. Neither of the child’s restriction fragments match those of possible father B 1 2 3 4 Starting position of sample

  33. Famous cases • In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father of her child Damien

  34. Famous Cases • Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. • He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

  35. Famous Cases • O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily on DNA evidence. • This case highlighted lab difficulties.

  36. Sequencing by Sanger Dideoxy Method • Controlled termination of replication • Uses 2’,3’ dideoxy analog of nucleotide

  37. Sequencing by Sanger Dideoxy Method

  38. Electrophoresis

  39. Fluorescence Detection

  40. Automated DNA Sequencing

  41. Southern blotting • Identification of restriction fragment

  42. Southern blotting • Identification of restriction fragment

  43. Research • Molecular biology

  44. Genetic Engineering

  45. Outline • Cell Transformation • Transforming Bacteria • Transforming Plant Cells • Transforming Animal Cells • Applications of Genetic Engineering • Transgenic Organisms • Cloning

  46. Introduction • Through genetic engineering scientists can combine DNA from different sources and this process is called “Recombinant DNA technology” • The secrets of DNA structure and functions have led to gene cloning and genetic engineering, manipulating the DNA of an organism

  47. Genetic Engineering • A set of techniques used to manipulate DNA in order to elicit a desired characteristic in the target organism • Recombinant DNA technology, or the creation of recombinant DNA, is a necessary component of genetic engineering

  48. Cutting DNA & Making Recombinant DNA • How Restriction enzymes work: • The Enzymes recognize specific sequences on Human and Bacterial Plasmids • The Enzymes cut the strands. • The cut produces DNA fragments with short strands on each end that are complementary to each other • “Sticky Ends” • Both the human DNA and the Plasmid “Open Up” with the same sticky ends remaining • They Bind Together

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