Manipulating genomes

• Define the terms "DNA sequencing", "terminator bases" and "high-throughput sequencing".

  DNA sequencing - working out the sequence of bases in a strand of DNATerminator bases/nucleotides- Specially created nucleotides using dideoxyribose which then prevent a new nucleotide joining and stops the new DNA strand growing. They are tagged with fluorescent dye - a different colour for each different base.High-throughput sequencing - new methods of sequencing DNA that are automated, very rapid and much cheaper than the original techniques.
• Describe the steps in DNA sequencing (the capillary method).

  DNA extracted from cells. Cells and their nuclei are broken down (either by grinding or enzymes) and the DNA is separated by precipitation and the proteins bound to it removed.DNA cut into fragments using restriction endonucleases. The fragments are spliced into vectors such as plasmids which are then introduced into bacteria. The bacteria reproduce, amplifying the plasmids with their introduced fragment.Fragments cut from plasmids using restriction endonucleases. Fragments heated, breaking the hydrogen bonds and creating single-stranded DNA.A primer is annealed to one end of the template strand.DNA TAQ polymerase and free nucleotides are added to create complementary strands. Some of these nucleotides are terminator bases made with dideoxyribose.As the reaction mix has many copies of the template strand, new complementary strands are made alongside each which will be different lengths according to which and when a terminator is added.The mixture is then heated, the new and template strands separate, more terminated strands are made. Cycle continues to create large number of terminated strands.The new strands are separated from parent strands by heating.The mix of fragments is loaded into one end of a capillary electrophoresis tube containing a gel. The strands are drawn along the tube with the smallest moving fastest - separating them based on length.At the far end the fragments pass through a laser beam which makes the dye on the terminal nucleotide fluoresce. This colour sequence is recorded and displayed by a computer to translate the colour into which base it refers to and therefore give the sequence.Computer analysis joins the fragments of data using overlapping sections to give the full sequence.
• Describe the reasons for developing new DNA sequencing technologies

  Faster, cheaper, less labour intensivePossible applications especially in medicine
• Define the terms "bioinformatics", "computational biology", "genome", "genomics", "proteomics", "DNA barcoding", "synthetic biology", and "epidemiology"

  Bioinformatics - the development of the software and computing tools needed to analyse and organise raw biological dataComputational biology - the study of biology using computational techniques to analyse large amounts of dataGenome - all of the genetic material of an organismGenomics - the branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes.Proteomics - the study an amino acid sequencing of an organisms entire protein complement.DNA barcoding - a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular speciesSynthetic biology - the design and construction of novel biological pathways, organisms or devices or the redesigning of existing natural biological systems.Epidemiology - the branch of medicine which deals with the incidence, distribution, and possible control of diseases and other factors relating to health
• Explain why new DNA sequencing methods are allowing genome-wide comparisons between individuals and between species

  New methods cheaper and faster. Computer analysis can highlight common patterns and differences.
• Explain why the comparison of many human genomes may help the understanding and treatment of human illness, and why "simple" answers very rarely occur

  Can compare those with conditions to those without looking for differences in genes that cause/increase susceptibility etc.Very few conditions caused by a change in a single gene - most are multiple genes in combination with environmental factors.
• Describe 4 reasons why scientists may want to analyse the genomes of pathogens

  Finding the source of an infectionIdentifying antibiotic resistant strains of bacteria so antibiotics only used when they will be effective.Track the progress of a disease outbreak and monitor potential epidemicsIdentify regions of the genome as potential targets for medicines and genetic markers for use in vaccines.
• Explain how DNA barcoding allows the identification of species

  Section of the genome is identified that is common to all species but varies between them so can be used to compare samples.
• Describe how DNA sequencing allows scientists to identify the evolutionary relationships between species

  DNA sequences compared and as the basic mutation rate can be calculated the time since divergence of the two species from a common ancestor can be calculated allowing evolutionary trees to be built up.
• Explain why, in theory, knowing a DNA sequence should allow you to identify the sequence of amino acids in the protein that the DNA sequence codes for. Also, explain why, in practice, this doesn't always provide the correct sequence of amino acids in the protein

  Specific codons code for specific amino acids so should be able to simply 'translate' the base sequence into an amino acid sequence.In practice introns are removed before translation and exons joined, sometimes in a number of different ways by spliceosomes to give functional mRNA. Post-translational modification also occurs that can alter the protein further from the original predicted amino acid sequence.
• Describe 4 techniques that could be classified as "synthetic biology". Describe the role of DNA sequencing in each technique.

  Genetic engineering - understanding which genes need to be modified requires an understanding of what they are and effective splicing etc. requires some knowledge of base sequences.Biological systems in industrial contexts such as immobilised enzymes etc. - genes for enzymes may be inserted into bacteria for reproduction etc.Synthesis of new genes to replace faulty genes - need to know the sequence that is desiredSynthesis of an entire new organism - creation of an artificial genome requires knowledge of the required base sequences.
• Define the terms "DNA profiling", "exon", "intron", "genome", "locus", "variable number tandem repeat" (VNTR), "minisatellite", "microsatellite", and "short tandem repeat" (STR).

  DNA profiling - producing an image of the patterns in non-coding DNA of an individualExon - a segment of a DNA or RNA molecule containing information coding for a protein or peptide sequenceIntron - regions of non-conding DNA or RNAGenome - all of the genetic material of an organismLocus - the positioin of a gene on a chromosomeVariable number tandem repeat (VNTR) - region where a short nucleotide sequence is organised as a tandem repeat (repetitions are directly adjacent to one another)Minisatellite - DNA segments located mainly near the ends of chromosomes that consist of repeating sequences of 20-50 base pairs repeated 50-100 times (also known as VNTRs)Microsatellite - a set of short repeated DNA sequences at a particular locus on a chromosome - 2-4 base pairs repeated 5-15 times (also known as STRs)Short tandem repeat (STR) - a set of short repeated DNA sequences at a particular locus on a chromosome - 2-4 base pairs repeated 5-15 times
• Describe how STRs vary and why they can be used to identify individuals

  Occur at the same position on chromosomes but the number of repeats varies between individuals so can be used to identify people.
• Name the 5 main stages in DNA profiling and describe the events in each stage.

  Extracting the DNA - PCR can be used to multiply the number of fragmentsDigesting the sample - the strands are cut into fragments using restriction endonucleases chosen to keep the satellites intactSeparating the DNA fragments - strands separated by electrophoresis then submerged in alkali to separate strands into single strands. Then transferred onto a membrane by southern blotting.Hybridisation - Radioactive or fluorescent DNA probes added in excess. Identify the microsatellite regions.Seeing the evidence - If radioactive x-ray images taken, if fluorescent put under UV light. A pattern of bars appears that is unique to each individual (except identical twins)
• Explain the role of restriction endonucleases in DNA profiling


  Cut the strands into fragments at specific nucleotide sequences.
• Suggest how DNA profiling can be used to answer questions about identity in different situations

  Forensic science - samples taken from crime scene matched to suspects/criminal databasesPaternity testing - to establish familial relationships in immigration casesIdentifying individuals at risk of certain diseases
• Describe how DNA can be amplified using the polymerase chain reaction (PCR)

  Short chains can be replicated so there are enough copies for testing/sequencing/analysis etc.DNA is heated to 95°C which breaks the hydrogen bonds holding the two strands together. Temperature is dropped to 55°C and primers added. These join or anneal to the complementary base sequence on the DNA strand.Temperature is raised to 72°C and Taq polymerase is added along with free nucleotides. The polymerase attaches to the primer and travels along the DNA strand, adding complementary nucleotides alongside the template strand. The result is two double stranded DNA molecules, identical to each other and the original molecule.The PCR process then restarts.
• Define the term "DNA primer".

  A short strand of DNA that serves as a starting point for DNA synthesis as polymerase can only add new nucleotides to existing strands of DNA.
• Explain the role of DNA primers, DNA nucleotides and DNA polymerase in PCR.


  Primers serve as a starting point for DNA synthesis as polymerase can only add new nucleotides to existing strands of DNA.DNA nucleotides are added so that they can be arranged according to base-pairing rules along existing strands and joined to form the new strands.Polymerase is the enxyme that joins nucleotides to form a strand.
• Explain the reason for the cycle of temperature changes in PCR

  95°C breaks the hydrogen bonds. The temperature must then be lowered (55°C) to allow the primer to form hydrogen bonds to anneal to tthe strand to be replicated. Taq polymerase then joins nucleotides on at 72°C, its optimum temp.
• Calculate how many DNA molecules will be produced after a specific number of PCR cycles

  2, 4, 8, 16 etc. - powers of 2
• Describe how gel electrophoresis can be used to separate DNA fragments of different length

  Restriction endonucleases are used to cut the DNA into fragments at a specific nucleotide sequence - creating fragments of different lengths. These are separated by size using electrophoresis. Fragments are negatively charged due to phosphate groups. Loaded into wells at one end of tank (agarose gel covered in buffer solution) tracking dye added so visible. Negative cathode attached to end with wells and anode to the other. Fragments attracted through gel towards anode. Smallest fragments move fastest. Result is visible bands. The pattern can be compared to others to see if same source etc. and/or a ladder of known lengths.
• Describe the two properties of DNA fragments that determine how far they travel in gel electrophoresis


  Distance depends on mass/length
• Define the term "DNA probe", explain the role of DNA probes in gel electrophoresis and describe how they can be labelled

  Fragment of DNA (or RNA) which is radioactively labelled and can be used to detect the presence of nucleotide sequences complementary to the sequence of the probe.Added to the DNA fragments - bind to complimentary sequence - 'hybridisation' - tagging them so a permanent image can be made of the banding pattern.
• Define the terms "genetic engineering", "transgenic", "vector", "recombinant DNA", "transformation", "electroporation", "marker gene", "DNA ligase" and "sticky ends"

  Genetic engineering - transferring a gene from one organism to another, usually of a different speciesTransgenic organism - an organism which has received a gene from another organismVector - a means of inserting DNA from one organism into the cells of another organismRecombinant DNA - DNA which has genetic material from two different organismsTransformation - the genetic alteration of a cell resulting from the direct uptake and incorporation of genetic material from its surroundings through the cell membrane(s)Electroporation - the use of very tiny electric current to transfer genetically engineered plasmids into bacteria or to get DNA fragments directly into eukaryotic cellsMarker gene - a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNADNA ligase - an enzyme that facilitates the joining of DNA strands together by catalysing the formation of a phosphodiester bondSticky ends - the ends of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other
• Describe the principles of genetic engineering.

  Isolating a gene for a desirable characteristic in one organism and placing it into another organism using a suitable vector
• Describe 2 ways in which a desired gene can be isolated

  DNA cut by restriction endonuclease - cuts at specific base sequence - either blunt or sticky ends (sticky preferred) ORmRNA extracted from a cell expressing the gene of interest - reverse transcriptase used to produce a strand of complimentary DNA. DNA polymerase then used to create the second strand.
• Explain how a plasmid can be used as a vector.

  Plasmid cut with same restriction endonuclease as gene to be inserted (so sticky ends match). Gene spliced into the plasmid using ligase.
• Explain how marker genes can be used to identify organisms that have been successfully genetically engineered

  Plasmids often genetically engineered to contain a marker gene e.g. antibiotic resistance. Gene of interest spliced into middle of marker gene so it ceases to function. If gene inserted, bacteria not resistant/don't fluoresce etc. so know which organisms contain recombinant DNA.
• Describe 4 ways in which a recombinant plasmid can be inserted into target cells

  Electroporation - brief electric shock applied to mixture of recipient cells and plasmids, temporarily disrupting membranes so plasmids can enterHeat shock - culture bacteria cells and plasmids in calcium rich solution and increase temp. - bacteria membrane becomes permeable and plasmids enterElectrofusion - tiny currents applied to membranes of two different cells fusing the cell and nuclear membranes to form a hybrid/polyploid cell containing DNA from bothTi plasmids - recombinant plasmids placed in the bacterium Agrobacterium tumefaciens which infects plant cells introducing the plasmid
• Describe how bacteriophages, DNA guns and liposomes can be used to insert DNA into cells

  Viral transfer - bacteriophages - viruses containing the gene attach to and introduce the gene. Virus chosen must be able to attach to receptors on the recipient cell.Ballistic impregnation - DNA gun fires tungsten or gold particles coated in DNA into plant cells using high velocity air jetLiposomes - DNA is wrapped in a hollow lipid sphere (liposome) which fuses with the cell membrane, introducing the DNA into the cell
• Describe how GM plant cells can be micro-propagated

  Once cells have been altered and all those not containing the recombinant DNA have been removed/killed/separated allow gene-altered cells to multiply to form a callus. Allow to grow shoots and roots and separate plants for transfer into soil to develop into fully differentiated adult plants. Process controlled through changing hormones within the growth medium.
• Describe the process of using genetic engineering to produce human insulin using bacteria

  mRNA extracted from Beta-cells and used to make double stranded DNA. Nucleotides added tothe ends to form sticky ends. VEctor used is a plasmid which has already been modified to contain genes leading to resistance to ampicillin and tetracycline. Plasmids cut by restriction endonuclease in middle of tetracycline resistance gene and insulin gene inserted using ligase. Plasmids put into bacteria ...A - Took up no plasmids - no resistanceB- Took up plasmids without insulin gene - resistant to bothC - Took up plasmids with insulin gene - only resistant to ampicillinSerial dilute bacteria and transfer to agar to grow colonies from single individuals. Test the resistance of some individuals form each colony to find those with insulin gene. Use these colonies to expand population in an incubator with optimum conditions, extract and purify insulin.
• Outline the positive and negative issues relating to the genetic manipulation of microorganisms

  Positive - used in industry and research Especially good for pure human medicines like insulin etc.Negative - pathogens could prove health risk. Ethical concerns over manipulation of pathogens to create biological weapons
• Describe the benefits of, and the ethical issues concerning, insect resistance in genetically modified soya beans.

  Benefits - no need to use pesticides to increase yield as crop already resistant Problems - non-pest insects and organisms that feed on insects may also be affected by toxins etc. Wider impacts on ecosystems.
• Name 7 useful characteristics that could potentially be genetically engineering into plants.

  Pest resistance, Disease resistance, Herbicide resistance, Extended Shelf-life, Growing conditions, Nutritional value, Medical uses
• Describe the benefits and risks of different genetically engineered characteristics in plants

  Pest resistance - no need to use pesticides to increase yield as crop already resistant, non-pest insects and organisms that feed on insects may also be affected by toxins etc. Wider impacts on ecosystemsDisease resistance - reduces crop losses/improves yield, genes transferring into wild plants and creating super weedsHerbicide resistance - herbicides can be used to reduce competition from weeds, lack of biodiversity and fear of superweedsExtended shelf-life - reduces food waste, may reduce commercial value and demandGrowing conditions - flood resistance, droughts etc.Nutritional values - extra vitamins etc. , people may be allergic to different proteins made in GM cropsMedical uses - plants could be used to produce human medicines and vaccines
• Describe the concerns people have over patenting and technology transfer in GM crops

  Poorer farmers often those who would benefit most but cannot afford to by the GM seeds from the companies. Also prevented from saving seed year to year so have to pay again each year.
• Describe 2 examples of genetically modified farm animals for food production

  Swine fever-resistant pigsFaster growing salmon
• Define the term "Pharming", and describe the two aspects to this field (including an example for each)

  The use of genetically modified animals to produce pharmaceuticals Creating animal models - for research e.g. knockout mice that are more likely to develop certain cancersCreating human proteins - Bacteria cannot produce all human proteins. Can insert human gene into sheep embryos (for example) along with promoter sequence so only expressed in mammary glands. Protein can then be harvested from milk.