Biotechnology Genomics PDF

BIOTECHNOLOGY GENOMICS 1  OVERVIEW Understanding how DNA works has allowed scientist to recombine DNA molecules and clone organisms. Biotechnology: application of tech process, invention or method to living organisms. Manipulation of DNA ha...

BIOTECHNOLOGY GENOMICS 1  OVERVIEW Understanding how DNA works has allowed scientist to recombine DNA molecules and clone organisms. Biotechnology: application of tech process, invention or method to living organisms. Manipulation of DNA has resulted in humans modifying bacteria to protect crops from pests or modifying said crops to produce higher yields. Biotechnologies have produced insulin, hormones, antibiotics and medicines. Comparative genomics gives new insights into relationship among species. DNA sequencing reveal genetic make-up. Manipulation of DNA poses social and ethical dilemmas as to what is right. 2  SELECTIVE BREEDING Selective Breeding: choosing organisms with wanted characteristics to mate for the purposes of making a desirable offspring. This method takes advantage of naturally occurring genetic variation to pass wanted traits to the next generation TEOSINTE INTERMEDIATES MODERN CORN 3  HYBRIDIZATION & INBREEDING Hybridization: crossing of dissimilar individuals to bring together most desirable trait from both parents Ex: disease resistance of one plant + great food-producing capacity of another Inbreeding: continued breeding of individuals with similar characteristics. Helps ensure characteristics that make each breed unique are preserved. Since most of the members of the breed are genetically similar, there is an increased chance that a cross will bring together two recessive alleles with a defect (sounds like…?) Less genetic variation also makes specie susceptible to emerging pathogen 4  MUTATION Mutation: heritable changes in DNA Can occur spontaneously, but can also be induced through the use of mutagens (radiation) Useful when applied to bacteria due to size and fast growth rate Bioremediation: use of microbes to remove pollutant from the environment ▶ Remove agricultural chemicals such as pesticides and fertilizers that leach from soil into groundwater, rivers, lakes and oceans. ▶ Toxic metals and oxides can also be removed from water because they are used as an energy source in the microbes metabolic processes. ▶ It has also been used in the cleanup of Oil Spills where bacteria can break it down 5  POLYPLOIDY Polyploid: organism that has incorrect number of chromosome sets Normally fatal on animals Can produce new species of plants that are larger and stronger than diploid relatives 6  HUMAN GENOME PROJECT International (US, UK, JAP, FR, GRM, CHN) scientific research project with the goal of determining the complete nucleotide sequence (+3 billion) of human DNA and map the location of every gene (20,000). Genome: an organism’s full set of genetic information contained within DNA It started in 1990 and was done (85%) in 2003. 99.7% done in May 2021 DNA Sequencing: Many companies will analyze samples of DNA which search for specific sequences to reveal ancestry or likelihood of an offspring developing certain genetic diseases Ethics: personal moral code. What an individual deems to be right or wrong. Should parents undergone required testing for genetic disorders? Does a couple have a right to produce children if they have high risk of genetic disorders? Does the government (insurance companies) play a role in eliminating costly disorders from population? 7 FUNCTION OF HUMAN GENES 8  DNA EXTRACTION Every cell contains DNA Easy to extract and isolate DNA for further manipulation Lysis buffer: solution of detergent used to split cells by breaking apart lipid molecules of membranes. Protease: enzyme that inactivates macromolecules by breaking down proteins Ribonuclease: (RNAse) break down RNA if no need to study gene expression. Cell debris is pelleted with a centrifuge. Supernatant is transfer to a clean tube. DNA is precipitated through the use of cold alcohol. DNA will be visible in form of gelatinous white mass. DNA can be stored at -80C Restriction enzymes are used to cut the extracted DNA into fragments 9  POLYMERASE CHAIN REACTION PCR: Technique used to amplify specific regions of the genome. Used for cloning gene fragments to analyze genetic diseases, identify contaminant foreign DNA in a sample, and amplifying DNA for sequencing. Insert DNA sample inside of a thermal cycler. Denature the template into single strands through the use of high temperature. Anneal primers to each original strand for new strand synthesis. Extend the new DNA strands from the primers with the use of Taq polymerase and added nucleotides. Repeat cycle copiously adding Taq polymerase as necessary. 10  GEL ELECTROPHORESIS Laboratory method used to separate mixtures of DNA, RNA or proteins according to their molecular size. The sample is stained with a dye and placed on an indentation or crevice (hole) called a well at one end of the gel (the beginning). The molecules travel through the gel at a speed that is inversely proportional to their lengths. In other words, small molecules will travel farther down the gel than longer/heavier molecules. In the end, stained bands can be observed which represent the different sizes of the molecules. Marker can be used to determine size of fragment 11  CHAIN TERMINATION/DIDEOXY METHOD Base sequence of fragments can be read to produce a completed genomic DNA Involves DNA replication of single-strand template with the use of a primer & deoxynucleotide (dNTP), a monomer of DNA. Primer & dNTP mixed with fluorescent labelled dideoxynucleotides (ddNTPs). ddNTPs are missing hydroxyl group (OH) at site where another nucleotide attaches to form a chain. Each ddNTP is labeled wit a different color of fluorophore helping to identify that position. Every time a ddNTP is incorporated into growing complementary strand, it terminates process of DNA replication. Each band of gel electrophoresis reflects size of DNA strand and the ddNTP that terminated the reaction. 12 CHAIN TERMINATION/DIDEOXY METHOD 13  NEXT-GENERATION SEQUENCING Since 2005 laboratories used automated techniques that are low-cost and can generate millions of short fragments in one day and use sophisticated software to get through process of organizing fragments in order. 1st genome to be completely sequences was that of a virus. (Why?) Model organism: specie studied to understand and compare it to the biological processes of similar organisms. Whole genome helps studies and apply to others. Escherichia coli (E.coli), Saccharomyces cerevisiae (most widely used yeast fermentator), Drosophila melanogaster (fruit fly), mouse Mus musculus, humans (Homo sapiens). 14  DATABASE Databases have been built for the use of the scientific community as a whole Worldwide information entered into GenBank at the National Center for Biotech Info (NCBI). Basic Local Alignment Search Tool (BLAST): software program that can compare sequences from GenBank and the alignments can construct phylogenetic trees. Whole genome-sequencing: process that determines the DNA sequence of an entire genome. Whole exome sequencing: lower cost alternative that only examines the exon coding region. 15  DNA MICROARRAYS DNA Microarray: method used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or silicon chip to ID active genes and sequences. Each cell has same genetic material, but genes are not active in every cell (Differentiation). Can study differences to understand which ones are directly correlated with what functions. Genomics working toward routinely using whole-genome sequencing to screen newborns (why?) for genetic abnormalities. Better methods to control microbes into production of important biotechnologies. Help clean up environment contaminants (sounds like…) Misuse could lead to discrimination based on person’s genetics and ethical concerns. 16  DNA MICROARRAYS DNA Microarray: method used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or silicon chip to ID active genes and sequences. Isolate mRNA from healthy cells Isolate mRNA from cancer cells Fluorescent label cancer (red) & normal (green) Probe will pick up color based on presence of each type of cell in each well. It can turn yellow in the presence of genes which are normal in both. This can help narrow down which sequences are defective to be targeted when technology is available. 17  RECOMBINANT DNA Recombinant DNA: plasmids with foreign DNA inserted into them that were created artificially (in lab), does not occur in nature. First observed by Griffith when R (rough) strain transformed into pathogenic (S) Smooth, despite the latter having been killed prior to mixing them Makes it possible to change the genetic composition of living organism Restriction enzyme cuts uneven (sticky ends) sequences which are brough back together like puzzles by DNA Ligase Genetic Markers: distinguish bacteria that carry plasmid from those that do not. Ex: antibiotic resistant genes to pick them out from general population 18  BACTERIAL TRANSFORMATION A 19  CRISPR CRISPR: Clustered regularly interspersed short palindromic repeats. Tool that could be used to directly change the DNA base sequence of a gene. Recombinant DNA molecule containing 2 genes is constructed: 1 gene codes for enzyme Cas9 and Other gene codes for RNA that will guide Cas9 enzyme to particular DNA sequence Guide RNA allows Cas9 enzyme to attach to DNA and cut both strands Enzymes repair broken ends by inserting random bases, which normally destroy function of gene. If single strand of DNA with bases matching the ones at break is injected into cell, then repair mechanism will use that one instead of random. This method has applications in Gene therapy: genetic engineering technique used to cure disease caused by mutated gene. 20  GENETIC ENGINEERING Genetic engineering: alteration of organism’s genotype using recombinant DNA to modify DNA and achieve desirable traits. Most common method involves addition of foreign DNA in form of recombinant DNA vectors generated by molecular cloning. Genetically modified organism: (GMO) organism that receives recombinant DNA. Transgenic: foreign DNA introduced into organism from a different species. Clone: population of genetically identical cells produced from a single cell. Unicellular: bacteria normally undergo this process as modified Mitosis (Binary Fission) Multicellular: A single cell from an adult organism is used to grow an entirely new individual that is genetically identical to the organism from which the cell was taken. 1st cloned animal was Dolly, a sheep in 1996 by Ian Wilmut. Nucleus of unfertilized egg cell is removed Egg cell is fused with donor cell containing nucleus from an adult Cell develops into embryo and implanted in uterus of foster mother 21  DOLLY A 22  DRAWBACKS Success rate of reproductive cloning was low at the time. Sheep have a lifespan of 10-12 years, Dolly lived 7 years. Speculation that because cell DNA belonged to older individual, it may affect life expectancy of cloned individual. Why? (T) Horses, bulls & goats have since been successfully cloned. Often exhibit physical abnormality. Why would we not want to find perfect individual & just clone them forever? Attempts at producing cloned human embryos as sources of embryonic stem cells for therapeutic purposes. Efforts met with resistance due to ethical considerations. 23  ANIMALS 30% of milk supply in US comes from cows injected with hormones to increase milk production Pigs can be modified to produce more lean meat or higher levels of omega fatty acids Farm raised salmon (AquaCulture) can be given transgenic growth-promoting hormones to grow faster than wild-caught. Mosquitos have been released in Florida, which are sterile to mate with those naturally present in environment to lower their numbers and prevent disease transmittal 24  AGRICULTURE Genetic modification has spread to food in order to make it less expensive, more nutritious and less harmful manufacturing process. Introduced in the US during 1996 make up a significant amount of current food supply. Could contain genes that produce proteins which make toxins harmless to humans, but that kill insects. The idea is to not have to spray the crops with pesticides and the added protection produces higher crop yields. Other resistances could be to viral infections and herbicides used to kill weeds. Currently developing resistances to rotting/spoiling Golden rice accumulated a vitamin absent in traditional white rice. 25  GENETICALLY MODIFIED FOODS Pros: Produce higher yield Reduce amount of land and energy devoted to agriculture Lower food cost and other plant-based products (cotton = clothes) Insect-resistant genes = less pesticide use, reducing chance of dangerous chemicals entering body or harm environment Scientific community has not produced any evidence of danger Cons: No long-term study into adverse effects have been conducted Loss of genetic diversity as farmers choose to only grow “best” version of crop Degree of uncertainty when it comes to animals Beneficial insects (pollinators) may also be affected 26  IDENTIFICATION With the exception of identical twins, humans have unique genomes DNA fingerprinting: analyzes sections of DNA that have little or no function, but vary widely from one individual to another. Sequences that contain genetic variation. Restriction enzymes used to cut DNA into fragments. PCR is used to amplify the sequence. Gel electrophoresis separates fragments based on size. Sequencing is used to identify DNA bases. Used in forensics (study of crime scene evidence) since 1980s. Used to identified fallen soldiers Used to establish paternity by matching alleles present in child, but not mother Y chromosome never undergoes recombination/crossover so father to son very similar Mitochondrial DNA passed directly from mother to child so easy to discern ancestry 27  ETHICS Ethics: moral principle that govern a person’s behavior In 2013 US Supreme Court ruled that genes found in nature cannot be patented. Patent: legal tool that gives indv. or company exclusive right to profit from innovations for a # of yrs However, altered or synthetic genes can be patented DNA can reveal private information: heritage, chances of developing certain diseases and used for evidence in criminal cases. Employee with access could racially profile Is personality linked with genes? If so, could you be discriminated against? Insurance companies can deny coverage because too expensive GMO products do not have a label identifying them. 28  ETHICS Ethics: moral principle that govern a person’s behavior Just because we have the tech to modify an organism’s characteristics, are we justified in doing so? We have the capability of manipulating human cells to cure disease. What is keeping us from engineering taller people, or changing eye color, hair texture, sex, blood group or appearance? What will be the consequences of cloning human beings, producing identical replicas? Could they be used to harvest organs? Ethics varies not only from country to country, but also amongst individuals present within the same location. In a democratic nation, all citizens, not just scientists, get to choose what is considered “right.” 29