21 - Manipulating genomes

Genome = all the genetic material the organism has Satellite DNA = within introns, telomeres and centromeres there are short sequences of DNA repeated many times Minisatellite or Variable Number Tandem Repeats (VNTR) = 20-50 base pairs that will be repeated 50-100s of times Microstellite or Short Tandem Repeats (STR) = 2-4 base pairs repeated 5-15 times Satellites appear on same position of the chromosomes but the number of repeats is unique to everyone except identical twins Producing a DNA profile is an image of the patterns in the DNA Producing a DNA profile Extracting the DNA DNA is extracted from a tissue sample Using Polymerase Chan Reaction (PCR) only a small fragment of tissue is needed Digesting the sample Strands of DNA cut into smaller fragments with restriction endonucleases Different restriction endonucleases have different recognition sites so cut the DNA at different sequences Restriction endonucleases give scientists the ability to cut the DNA strands at specific introns Use a mixture of restriction enzymes to leave repeating units intact so the fragments at the end are a mix of micro- and minisatellite regions Separating the DNA fragments Cut fragments of DNA are separated through electrophoresis (technique that uses the negative charge of DNA to move it through a gel medium and separate the strands by length) The gel is then immersed in an alkali solution to separate the DNA strands from one another The single-stranded DNA fragments are then transferred onto a membrane by Southern blotting Hybridisation  Radioactive or fluorescent DNA probes (short DNA or RNA sequences complementary to known DNA fragments) are added in excess to the DNA fragments on the membrane They bind to complementary strands of DNA in the right pH and temperature The probes identify microsatellite regions that are more varied than the larger minisatellite regions Excess probes are washed off Seeing the evidence If radioactive probes were added, x-ray images are taken of the membrane If fluorescent probes were added, the membrane is placed under a UV light so they glow <-- most commonly used  Fragments give a pattern of bars (the DNA profile) which is unique to everyone Electrophoresis DNA fragments put into wells in agarose gel strips which contain a pH buffer solution One or two wells have a DNA fragment with a known length to compare the others to Electric current is passed over the electrophoresis plate DNA fragments at cathode end travel toward the anode end Rate of movement depends of the length of the DNA fragment (smaller move quicker and further) Current switched off when shortest fragments reach the anode Gel is placed in alkali solution to denature the DNA fragments Southern blotting --> strands are transferred to a nitrocellulose paper or nylon membrane which is placed over the gel Membrane covered with several sheets of blotting paper to remove the alkali solution by capillary action Polymerase Chain Reaction (PCR) DNA sample can be amplified if put into a PCR machine with an excess of nucleotide bases, small primer DNA sequences and DNA polymerase The temperature is increased to 90-95C for 30 seconds to denature the DNA (break the H bonds) The temperature is decreased to 55-60C so that the primers anneal to the DNA fragments The temperature is increased to 72-75C for a minute (optimum temp for enzyme)  DNA polymerase builds up the complementary strand by binding the free nucleotides to the original sequence Used of DNA profiling Forensic science --> DNA from a crime scene amplified and used to check suspects & added to the police database Prove paternity of child  Used in immigration cases to prove or disprove family relationships Used to demonstrate evolutionary relationships between species Indentification of people at risk of developing particular diseases which are associated with certain microsatellite patterns

The whole human genome was sequenced and published in 2003 Principles of DNA sequencing (Sanger method) DNA chopped into fragments with restriction enzymes Terminator bases (thymine, adenine, cytosine and guanine) that are modified are used Each base has a fluorescent tag attached - A = green, G = yellow, T = red and C = blue The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of free nucleotides and the terminator bases Mixture is put into a thermal cycler (basically PCR - 96C DNA strands separate, 50C primers anneal, 60C the DNA polymerase builds up the complementary DNA strands) Each time a terminator base is randomly used, the synthesis of DNA is stopped so no more bases are added --> results in many DNA fragments with different lengths After many cycles, all the possible DNA chains will be produced The DNA fragments are then separated according to their length by capillary sequencing (gel electrophoresis in capillary tubes) Lasers detect the different colours of the terminator bases and therefore the order of the sequence This order of bases is the complementary strand to the original DNA fragments so can be used to work out the original easily The data from the sequencing is fed into a computer that reassembles the genome by comparing all the fragments and finding areas that overlap Once the genome is assembled you can identify the genes that code for specific characteristics Next-generation sequencing Techniques have become faster and more automated with time Instead of using gel or capillary tubes, the reaction takes place on a plastic slide now Millions of fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments All the clusters are sequenced and imaged at the same time so it's known as the massively parallel sequencing or next-generation sequencing  ^ constantly being refined and developed  Enables us to use the information from the genome in many new and different ways

Computational biology and bioinformatics Bioinformatics = development of software and computing tools to organise and analyse raw biological data  Computational biology = uses the data from bioinformatics to build theoretical models of biological systems to predict what will happen in certain circumstances (study of biology using computational techniques --> work out 3D structures of proteins or understanding molecular pathways like gene regulation) Genome-wide comparisons Genomics = field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes Analysing the human genome Computers can analyse and compare the genomes of many individuals, revealing patterns of DNA we inherit and the diseases to which we are vulnerable ^ enormous implications for health management and the field of medicine But scientists keep in mind that with the exception of a few genetic diseases, the interaction of our genes and the environment are what influence our physiology  Analysing the genomes of pathogens Enables doctors to find out the source of an infection Lets doctors identify antibiotic-resistant strains of bacteria so antibiotics will only be used when they'll be effective --> stops spread of antibiotic resistance Scientists can track the progress of an outbreak of a potentially serious disease or manage epidemics Identify regions of the genome of pathogens that could be useful targets in the development of new drugs & identify gene markers to use in vaccines Identifying species (DNA barcoding) Genome analysis provides another way to identify species --> compare a sample against a standard sample for the species One useful technique is to identify particular sections of the genome that are common to all species but vary between them so comparisons can be made <-- DNA barcoding For animals, they use 648 base-pairs from mitochondrial DNA in the gene for enzymes involved in cellular respiration ^ section is small enough to be sequenced quickly and cheaply yet varies between different species In plants, two regions of the DNA of the chloroplasts are used instead Barcoding system isn't perfect - scientists have not come up with suitable DNA regions to use for fungi or bacteria Searching for evolutionary relationships DNA sequences of different organisms can be compared and the basic mutation rate can be calculated to see how long ago the species diverged from their common ancestor Genomics and proteomics Proteomics = study of amino acid sequencing of an organism's entire protein complement Has revealed that some genes can code for many different proteins because the expected phenotype of an organism based on its sequenced genome is different to the actual phenotype       Spliceosomes Spliceosomes = enzyme complexes that join the exons to be translated together They may join some exons in a variety of ways therefore a single gene may produce several versions of mature mRNA which all code for different arrangements of amino acids giving different proteins and therefore different phenotypes       Protein modificiation A protein coded for by a gene may remain intact or it may be shortened or lengthened to give a variety of other proteins Synthetic biology Synthetic biology = the design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems Different techniques: Genetic engineering --> may involve a single change in a biological pathway or relatively major genetic modification of an entire organisms Use of biological systems or parts of biological systems in industrial complexes (fixed or immobilised enzymes) The synthesis of new genes to replace faulty genes (cystic fibrosis)  The synthesis of an entire new organism

Isolated the desired gene Most common technique is the use of restriction endonucleases to cut the required gene from the DNA Some make a clean, blunt-ended cut in the DNA but others cut the DNA strands unevenly leaving sticky ends Sticky ends make it easier to insert the desired gene into the DNA of a different organism Another technique involves isolating the mRNA for the desired gene and using reverse transcriptase to produce a single strand of complementary DNA ^^ easier to identify the desired gene The formation of recombinant DNA Vectors Most common vectors are bacterial plasmids (small circular molecules of DNA that can replicate independently) Once the plasmid gets into a host cell it can combine with the host DNA to form recombinant DNA The plasmids used as vectors are often chosen because they contain a marker gene which allow the scientists to see when the plasmid has been taken up The same restriction endonuclease is used to cut open the DNA of the plasmid so that the sticky ends of the isolated gene will match up with the hole Once the isolated gene and plasmid DNA have matched up by complementary base pairing, DNA ligase forms the phosphodiester bonds to join the DNA strands together A second marker is often added alongside the desired gene If the DNA is inserted successfully, the additional marker gene won't function (if its a fluorescent one then the bacterium will not fluoresce if its been engineered successfully) Transferring the vector Transformation = transfer of the plasmid with the recombinant DNA into the host cell One way is to culture the bacterial cells in a calcium-rich solution at a high temperature --> increases the permeability of bacterial membranes so the plasmids enter Another is electroporation where a small electric current is applied to the bacteria to make the membranes porous and allow the plasmids in ^^ can be used to get DNA fragments directly into eukaryotic cells (new DNA will pass through cell and nuclear membrane to fuse with the nuclear DNA) ^^^ effective but cannot be 100% controlled and damages the membrane permantently        Electrofusion Tiny electric currents applied to membranes of two different cells Fuses cell and nuclear membranes of the two cells to form a hybrid or polyploid cell with DNA from both Used to produce GM plants Not used for animal cells --> different membrane structures Important in the production of monoclonal antibodies --> combination of a plasma cell and a tumour cell Used to identify pathogens in both plants and animals and in treatment of cancers Engineering in different organisms Prokaryotes Genetically modified to produce substances that are useful to humans like hormones. clotting factors, antibiotics, pure vaccines and enzymes for industry Plants Use of bacterium that causes tumours in healthy plants Desired gene (e.g. pesticide production) placed in the plasmid of the bacterium with a marker gene (for antibiotic resistance or fluorescence)  This is the carried directly into the plant cell DNA Transgenic cells form a callus (mass of GM plant cells) each of which can be grown into a new GM plant OR by electrofusion --> cells produced are polyploid (have chromosomes from both original cells) Removal of plant cell walls by cellulases Electrofusion to form a new polyploid cell Use of plant hormones to stimulate growth of new cell wall Callus formation Production of many transgenic plants Animals Harder to modify animal cells Membranes less easy to manipulate Important technique to allow animal cells to make medically important proteins and to try and cure genetic diseases

Genetic manipulation of microorganisms GM bacteria used to create large quantities of useful substances Used to store a living record of the DNA of another organism in DNA libraries <-- source of DNA fragments for further genetic engineering applications Widely used tool in research for developing medical techniques and industrial processes Genetically engineered pathogens not as widely used for health and safety risks --> used only for medical research and is strictly regulated Ethical concerns Relatively little ethical debate over GM microorganims except for the manipulation of pathogens for biological warfare GM plants Help feed the ever-growing population Overcome environmental issues     Insect resistance in GM soya beans Major world crop Scientists inserted a gene to make them resistant to pest insects and to resist weed killer Farmers can spray fields with weedkiller without killing the soya beans --> reduce competition  Farmers don't need to use pesticides Enables bigger yields with less labour and less expense       Benefits and risks of GM crops Pest resistance --> reduce amount of pesticide spraying so protects the environment and helps farmers, higher yields , non-pest insects may be damaged by the toxins, pest may become resistant Disease resistance --> crop varieties resistant to common diseases to reduce loss/increase yield, genes may spread to wild populations and cause problems Herbicide resistance --> herbicides can be used to reduce competition and increase yields, biodiversity reduced, fear of superweeds Extended shelf-life --> reduces food waste, reduce commercial value and demand for the crop Growing conditions --> plants can grow in wider range of conditions Nutritional value --> value of crops can be increased, people may be allergic to the different proteins Medical uses --> plants may be used to produce human medicines       Patenting and technology transfer  People in less economically developed countries prevented from using GM plants because patents make them too expensive GM animals Harder to produce GM vertebrates Scientists researching use of microinjections and modified viruses to carry new genes to animal DNA Goals include the transfer of disease resistance or to modify the physiology of farm animals (swine-fever resistant pigs or faster-growing salmon)       Pharming Pharming = genetic engineering of animals to produce human medicines Creating animal models --> addition or removal of specific genes so that animals develop certain diseases for testing of new therapies Creating human proteins --> introduction of human gene coding for medically required protein, gene is inserted into pregnant cow, sheep or goat so that the gene is expressed and the protein is present in their milk       Ethical issues Should animals be genetically engineered to be tested on? Is it right to put human genes into animals? Are we certain it doesn't cause harm? Does modifiyng animals reduced them as commodities? Is the animals' welfare compromised? Gene therapy in humans Some human diseases are the result of faulty genes Somatic gene therapy = replacing the mutant allele with a healthy allele in the affected somatic cells Only a temporary solution --> the somatic cells are replaced by stem cells which only have the faulty allele before long Treatment won't stop the faulty gene being passed on to any children the individual has Germ line gene therapy = inserting of healthy allele into egg or embryo immediately after fertilisation (IVF) Illegal for human embryos Potential impact on human embryos is unknown Human rights of unborn baby could be seen as being violated Technology may eventually be used to let people choose desirable characteristics for their offspring