* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 7. Biotechnology- Using Molecular Biology and Genetic Engineering
Comparative genomic hybridization wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Gel electrophoresis wikipedia , lookup
List of types of proteins wikipedia , lookup
Molecular evolution wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Restriction enzyme wikipedia , lookup
Genomic library wikipedia , lookup
Non-coding DNA wikipedia , lookup
DNA vaccination wikipedia , lookup
DNA supercoil wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Molecular cloning wikipedia , lookup
Biotechnology Introduction Biotechnology is essentially the use of living organisms and their products for health, social or economic purposes. Biotechnology is widely considered to be the growth technology of the 21st century which will lead to huge growth in the Biotechnology industry and exciting opportunities for graduates 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. 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 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. 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 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. 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! Hybridization Louis Burbank was the greatest selective breeder of all time. He developed the disease-resistant potato and more than 800 varieties of plants. 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 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 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 Increasing Variation Breeders increase the mutation rate by using radiation and chemicals Molecular Biology 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 Outline Restriction-enzyme analysis Gel electrophoresis The polymerase chain reaction (PCR) DNA Fingerprinting DNA sequencing Blotting techniques Recombinant DNA Restriction Enzyme Analysis aka restriction endonucleases Recognizes specific base sequences and cleave the nucleic acid PALINDROMES Two-fold rotational symmetry Generates fragments of DNA 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 Gel Electrophoresis A technique for separating DNA (or protein) molecules on the basis of size. DNA plus restriction enzyme Power source Longer fragments Mixture of DNA fragments Gel Shorter fragments Gel Electrophoresis Utilizes agarose or polyacrylamide gels for the separation of DNA DNA plus restriction enzyme Power source Longer fragments Mixture of DNA fragments Gel Shorter fragments Electrophoresis Electrophoresis • Electrophoresis is used to map the structure of a DNA fragment Electrophoresis Stained gel result Visualization may be achieved through UV dyes or radioactive agents 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) PCR Requirements Template (genomic DNA or cDNA population) Oligonucleotide primers DNA polymerase (Taq polymerase) dNTP Thermal cycler Cycles (25 – 30) Denaturation 95o C Separate strands DNA Synthesis 70 – 75 oC (ideal temp for Taq polymerase) Thermus aquaticus (Taq) Annealing 50 – 70 oC (~5o C lower than Tm) 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 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 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 Steps: 1. Get DNA sample 2. Amplify with PCR 3. Cut with restriction enzyme 4. Run resulting fragments on gel electrophoresis 5. Analyze result A sample with the shorter DNA fragments travels through the gel faster than a sample with the larger fragments V S S1 S2 S3 DNA profiles 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 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. 1 2 3 4 Starting position of sample Neither of the child’s restriction fragments match those of possible father B Famous cases In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father of her child Damien 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. 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. Sequencing by Sanger Dideoxy Method Controlled termination of replication Uses 2’,3’ dideoxy analog of nucleotide Sequencing by Sanger Dideoxy Method Electrophoresis Fluorescence Detection Automated DNA Sequencing Southern blotting Identification of restriction fragment Southern blotting Identification of restriction fragment Research Molecular biology Genetic Engineering Outline Cell Transformation Transforming Bacteria Transforming Plant Cells Transforming Animal Cells Applications of Genetic Engineering Transgenic Organisms Cloning 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 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 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 Restriction enzyme cleaving Recognition sequences DNA sequence Recognition sequences DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end Vectors = carriers of DNA fragments Plasmids Naturally occurring circular, double-stranded DNA that act as accessory chromosomes in bacteria; DNA fragments (15, 000 bp) λ phage Bacteriophage (virus) DNA; for larger DNA fragments (23, 000 bp) Yeast and bacterial artificial chromosome Laboratory-designed carriers for larger DNA fragments Confirmation of a Cloned Gene Southern Blot can be used to identify a specific gene: 1. Cut DNA from bacteria with restriction enzymes. 2. DNA fragments are separated by a gel soaked in a chemical solution. Confirmation of a Cloned Gene 3. The DNA separated is then transferred to a membrane (blotted) and a probe solution is added. Probes: radioactive RNA or single-stranded DNA pieces that are complementary to the gene of interest 4. Only DNA fragments complementary to the probe will form and bind bands Producing Recombinant Bacteria 1. Remove bacterial DNA (plasmid). 2. Cut the Bacterial DNA with “restriction enzymes”. 3. Cut the DNA from another organism with “restriction enzymes”. 4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria. 5. Reproduce the recombinant bacteria. 6. The foreign genes will be expressed in the bacteria. When a bacteria or other cell takes in a foreign piece of DNA such as a plasmid, the process is called transformation If transformation is successful, the recombinant DNA is integrated into one of the chromosomes of the cell. Creating HGH Recombinant DNA Gene for human growth hormone Gene for human growth hormone Human Cell Sticky ends Bacterial Cell Bacterial chromosome Plasmid DNA recombination DNA insertion Bacterial cell for containing gene for human growth hormone What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology Some Benefits of Recombinant Bacteria 1. Bacteria can make human insulin or human growth hormone. 1. Bacteria can be engineered to “eat” oil spills. Genetically Engineered Drugs and Vaccines Today, many pharmaceutical companies around the world produce important proteins using genetic engineering. Vaccine: a solution containing all or part of a harmless version of a pathogen; used to prevent viral diseases (don’t respond to drugs) Many vaccines are made using genetic engineering The DNA of plants and animals can also be altered. PLANTS 1. disease-resistant and insect-resistant crops 2. Hardier fruit 3. 70-75% of food in supermarket is genetically modified. Improving Crops Genetic engineers can add favorable characteristics to a plant Plants become resistant to insects (no longer need pesticides); resistant to weed killer (so crops won’t die, but weeds will); improved nutrition – rice + corn Pest Resistance: Bt Corn Plant Transformation Gene to be transferred Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome Agrobacterium tumefaciens Cellular DNA Recombinant plasmid Plant cell colonies Complete plant is generated from transformed cell Transformed bacteria introduce plasmids into plant cells How to Create a Genetically Modified Plant 1.Create recombinant bacteria with desired gene. 2. Allow the bacteria to “infect" the plant cells. 3. Desired gene is inserted into plant chromosomes. Genetically modified animals are called transgenic animals. TRANSGENIC ANIMALS 1. Mice – used to study human immune system 2. Chickens – more resistant to infections 3. Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk Transgenic Goat Human DNA in a Goat Cell . This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk. Transgenic Cows Growth hormones are given to cows to produce more milk Human genes are added to farm animals in order to have human proteins in their milk The human proteins are extracted from milk and sold to pharmacy companies. Useful for complex proteins that can’t be made in bacteria Transgenic animals How it works: an intact nucleus from an embryonic cell (whose DNA has recombined with a human gene) is placed into an egg whose nucleus has been removed. The “new” egg is then placed into the uterus of an animal. How to Create a Transgenic Animal Animal Cloning A clone is a member of a population of genetically identical cells produced from a single cell The goal of cloning is to mass produce a certain individual with desired characteristics No need for breeding, and the desired characteristics can be preserved. How it works: an intact nucleus from a cell is removed Cloning Animals The nucleus is fused with a egg cell (whose nucleus has been removed) taken from another adult The fused cell begins to divide and the embryo is placed in the uterus of a foster mother. The “new” egg is then develops normally. Cloning A donor cell is taken from a sheep’s udder. Donor Nucleus Egg Cell These two cells are fused using an electric shock. Fused Cell The nucleus of the egg cell is removed. The fused cell begins dividing normally. Cloned Lamb An egg cell is taken from an adult female sheep. The embryo is placed in the uterus of a foster mother. Embryo The embryo develops normally into a lamb— Dolly Foster Mother PERPETUATE: A pet cloning company A company founded in 1998 by Dr. Heather Bessoff and Ron Gillespie “PERPETUATE's packaged service provides the pet owner with the following three biotechnologies performed on their pet's cell biopsy” Cell culturing, DNA preservation, Genotyping.