Manipulating Genomes

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  • Manipulating Genomes
    • DNA Profiling
      • The human Genome
        • The genome of an organism is all of the genetic material that it contains
        • Exons are coding DNA and introns are non-coding DNA
        • Satellite DNA is DNA that can be found in repeating short sequences within introns, telomeres and centromeres,
        • In a mini-satellite 20-50 base pairs can be repeated 50+ times
        • In a micro-satellite 2-4 basses are repeated 5-15 times
        • Satellites always appear in the same places on chromosomes but the number of repeats of each mini or micro are varied between individuals.
      • Producing a DNA profile
        • 1) Extraction- DNA is extracted from the sample
        • 2) Digestion- restriction endo-nucleases cut the DNA into fragments
        • 3) Separation- Fragments are separated using gel electro-phoresis, DNA fragments are then transferred  from the gel to nylon membrane in Southern Blotting
        • 4) Hybridisation- DNA probes are added to label the fragments, these radioactive probes attach to specific fragments
        • 5) Development- membrane wit radioactively labelled DNA fragments is placed onto an X-ray film. Development of the X-ray film reveals dark bands where the radioactive or fluorescent DNA probes have attached.
      • Polymerase Chain Reaction (PCR)
        • PCR is a version of the natural way by which DNA is replicated
        • The following are placed in a vial in a PCR machine: DNA sample, excess A, T, C and G, small primer DNA sequences and DNA polymerase
        • There is careful control over the temperature within the PCR machine. It changes rapidly at programmed intervals. This triggers different stages of the process.
        • The reaction can be repeated many times by the PCR machine.
        • About 30 repeats gives more than enough copies of the original DNA sample for DNA profiling to happen.
        • Main stages of the PCR
          • 1) The DNA fragment is separated into strands. Occurs at 95 degrees, this denatures the H bonds between the strands
          • 2) Temp is decreased to 55 degrees, the DNA primers are annealed
          • 3) Temp is increased to 72 degrees this is the optimum temp for DNA polymerase, DNA polymerase adds bases to the primer creating complimentary strands of DNA., this makes double stranded DNA which is identical to the original sequence.
      • Uses
        • Best known for its use in forensic science
        • It is able to provide evidence for the innocence or guilt of a suspect
        • Used to prove the paternity of a child
        • Used in immigration to prove or disprove family relationships.
        • Used to identify the species to which an organism belongs
        • Identifying individuals who are at risk of developing particular diseases.
    • DNA sequencing and analysis
      • The Human genome
        • In 1990 the Human Genome Project (HGP) was established.
        • International project where scientists from different countries worked to map the entire human genome.
        • The first complete human genome was published in 2003
      • Principles of DNA sequencing
        • 1) DNA to be sequenced is mixed with a primer, DNA polymerase and excess of normal nucleotides and terminator bases
        • 2) The mixture is placed in a thermal cycler (same as used in PCR), at 96 degrees the double stranded DNA separates into single strands, at 50 degrees the primers anneal to the strand.
        • 3) At 60 degrees DNA polymerase starts to build up a new DNA  by adding complimentary nucleotides
        • 4) Each time a terminator base s incorporated instead of a normal nucleotide the synthesis is terminated and no more bases can be added. The many fragments will be different lengths, after many cycles the DNA fragments are separated by their length through capillary sequencing
          • Capillary sequencing works like gel electrophoresis through capillary tubes, the fluorescent markers on termiantor bases are used to identify the final base on each fragment, Lasers detect the different colours and so the order of the sequence,
        • 5) The order of bases in the capillary tubes show the sequence of the new strand, the data from the sequencing process is fed into a computer,this can be used to work out the original DNA sequence.
      • Next generation sequencing
        • Technological developments have led to new automated, high-throughput sequencing processes.
        • Instead of a gel or capillary tubes, the sequencing takes places on a flow cell
        • All clusters are sequenced and imaged at the same time.
        • Clusters of identical DNA fragments are formed from in -situ replication by PCR
        • It has meant that the entire human genome can be sequenced in a few days and that of a bacterium in 24 hours.
    • Using DNA sequencing
      • Bioinformatics is the development of the software and computing tools needed to organise and analyse raw biological data.
      • Computational biology then uses the data from bioinformatics to build theoretical models of biological systems, these can be used to predict what will happen in different circumstances.
      • Computational biology is the study of biological using computational techniques especially in the analysis of large amount of bio-data.
      • Genomics is the field of genetics which applies DNA sequencing methods and computational biology to analyse the structure and function of genomes.
      • Computers can analyse and compare the genomes of many individuals this reveals patterns in the DNA we inherit and the diseases for which we are vulnerable.
      • Analysing genomes of pathogens. Sequencing the genomes of pathogens has become relatively cheap, this enables:
        • Doctors to find out the source of an infection
        • Doctors to identify antibiotic resistant strains of bacteria.
        • Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics.
        • Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines.
      • Identify species (DNA bar-coding)
        • Scientists identify species using relatively short sections of DNA from a conserved region of the genome.
        • The section is small enough to to be sequenced cheaply and quickly.
        • The bar-coding system is not perfect as so far scientists have not come up with suitable regions for fungi and bacteria.
        • Has a big impact on classification
      • Genomics and Proteonomics
        • Proteonomics is the study and amino acid sequencing of an organisms entire protein compliment.
        • In theory the DNA sequence of the genome should enable you to predict the sequence of amino acids in all of the proteins that it makes.
        • Spliceosomes
          • mRNA transcribed from the DNA in the nucleus contains both introns and exons, before it is translated it is modified in a number of ways:
            • Introns are removed, then the exons are joined together by enzyme complexes known as spliceosomes.
            • Spliceosomes may join the same exons in a variety of ways, this means that a single gene may produce several different versions of mRNA.
            • This would code for different arrangements of amino acids, giving different proteins and causing several different phenotypes.
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        • Protein Modification
          • The study of proteonomics is giving us increasing knowledge of the extremely complex relationship between the genotype and phenotype.
      • Synthetic Biology
        • Synthetic biology is 'an emerging area of research that can broadly be described as the design and construction of novel artificial biological pathways, organisms or devices, or the redesign of existing natural biological systems.'
        • It includes many different techniques such as:
          • Genetic Engineering- can involve a single change in a biological pathway or relatively major genetic modification of an entire organism
          • Use of biological systems or parts of biological systems in industrialised contexts.
          • Synthesis of new genes to replace faulty genes
          • The synthesis of an entire new organism.
    • Genetic Engineering
      • The first stage of  genetic modification is to isolate the desirable gene
        • The common technique involves the use of enzymes called restriction endonucleases, these cut the required gene from the DNA of an organism.
          • Some make a clean cut but most cut unevenly, this leaves one of the strands of the DNA fragment a few bases longer than the other strand.
          • These unpaired regions are called sticky ends.
          • Sticky ends make it much easier for the desired gene to be inserted into the DNA of another organism.
        • Another technique is to isolate the mRNA and using reverse transcriptase a single strand of complimentary DNA can be made
          • The advantages of this are that it is easier to identify the desired gene .
      • Formation of recombinant DNA
        • Vectors
          • The most commonly used vectors in genetic engineering are bacterial plasmids.
            • Once a plasmid gets into a new host cell it can combine with the host DNA to form recombinant DNA
            • Plasmids are particularly effective in the formation of genetically engineered bacteria used.
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            • Plasmids which are used as vectors are chosen usually because they have a marker gene.
            • To insert a DNA fragment into a plasmid it must first be cut open, by restriction endonuclease This creates complimentary sticky ends.
              • Once the complimentary bases of the two sticky ends are lined up DNA ligase forms phospho-diester bonds between sugar and phosphate groups on the two strands of DNA, this joins them.
            • Plasmids used as vectors are usually given a second marker gene, this is used to show that the plasmid contains the recombinant gene. The marker gene is usually put into the plasmid via genetic engineering
              • The Plasmid is then cut by a restriction enzyme within the marker gene to insert the desired gene. If the DNA fragment is inserted successful the marker gene won't function.
            • The Plasmid is then cut by a restriction enzyme within the marker gene to insert the desired gene. If the DNA fragment is inserted successful the marker gene won't function.
        • Transferring the vectors
          • Plasmid with recombinant DNA must be transferred into the host cell in a process called transformation.
            • One process is to culture the bacterial cells and plasmids in a calcium rich solution and to increase the temperature, this makes the bacterial membrane becomes permeable and the plasmids can enter
            • Electro-poration is another method. A small electrical current is a[[lied to the bacteria making the membranes more porous, plasmids will then move into the cells.
              • Electroporation can also be used to get DNA fragments directly into eukaryotic cells, the new DNA will pass through the cell membrane and the nuclear membrane to fuse with the DNA.
          • Electrofusion
            • Tiny electrical currents are applied to the membranes of two different cells, this fuses the cell and nuclear membranes of two different cells together to form a hybrid polyploid cell, this will contain DNA from both original cells.
            • Used in the GM of plants as it is harder to do for GM of animals due to having different membranes with different properties.
            • Important in the production of monoclonal antibodies, a monoclonal antibody is made by a combination of a cell producing a single type of antibody with a tumour cell, this means that it divides rapidly in cukture,
              • Monoclonal antibodies are now used to identify pathogens in both animals and plants, and in the treatment of a number of diseases.
    • Gene Technology and Ethics
      • GM microorganisms are a widely used tool in research for developing novel medical treatments and industrial processes.
      • GM pathogens are not widely used in these applications for the health and safety of the researchers and wider public
        • One of the issues with GM pathogens is that they could lead to some kind of biological warfare.
      • GM crops
        • Pros: increased yield, reduce pesticide spraying, disease resistant varieties can be made and there can be a reduction in competing weeds etc.
        • Cons: could be toxins, insects/ pests could become resistant over time, transferred genes might spread to wild type, reduce biodiversity and fear of super weeds.
      • Pharming is the use of genetic engineering in animals to form human medicines.
      • Gene Therapy
        • Somatic Cell Gene Therapy
          • Replacing the mutant allele with a healthy allele in the affected somatic (body cell)
          • A treated individual can still pass the faulty allele onto their children
        • Germ Line Cell Gene Therapy
          • Inserting a healthy allele into the germ cells (usually the eggs) or into an embryo immediately after fertilisation.
          • The individual would be born healthy with the normal allele in place and would pass it onto their own offspring
          • A major ethical issue with this is that it would lead to designer babies.
        • A major ethical issue with this is that it could be used in the future to enable people to choose desirable or cosmetic characteristics of their offspring.

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