Presentation on theme: "Genomes and Gene technologies"— Presentation transcript:
1Genomes and Gene technologies F215 control, genomes and environmentModule 2 – Biotechnology and gene technologies
2Learning OutcomesOutline the steps involved in sequencing the genome of an organism.
3Genomes 1950’s Gene technology Developing rapidly Learnt that DNA is the genetic materialGene technologyUse of DNA to produce something that we wantDeveloping rapidlyBecoming more and more able to alter genes within organismsPoint to think aboutJust because we can do something does that mean that we should do it?
4Manipulating DNA Advances in DNA technology DNA profiling (genetic fingerprinting)Genomic sequencingComparative genome mappingGenetic engineeringGene therapy
5GenomeAll the genes possessed by an individual organism, or a population of organisms.The whole sequence of bases in all of the DNA in an organism.
6Human Genome Project1988International project started to discover the sequence of bases in each of the 23 different types of chromosomes found in human cells2000A working draft sequence was produced
7Facts about human genome 99.9% of the base sequence in our DNA seems to be identical in all humansVariation is caused by the variable 0.1%This 0.1% is very variableVariations can be used for DNA profiling2% of human genome codes for the manufacture of proteinsGiving around genes in the human genome (even mice have more!!)The rest of the “junk” genome, may be involved in gene expression
8Sequencing a genome The genome is broken up and sequenced in sections Sequencing is carried out on overlapping regionsStagesGenome mappingMechanically break into smaller sectionsCarry out sequencing on overlapping sectionsAnalyse and put back together to form the complete code
9Sequencing a genome Look at the worksheet “sequencing a plant gene”Make multiple copies of the genome using PCRDNA randomly broken up into lengths 2000bp – 10000bp longThese lengths can then be broken up further
10Sequencing a genomeMake multiple labelled copies of each small length of DNALengths of DNA mixed withDNA PolymerasePrimer“normal” DNA nucleotides“labelled” DNA nucleotidesdideoxy nucleotidesFour colours of dye used for bases A, T,G and CIf incorporated in nucleotide chain – chain stops growing
11Sequencing a genome Result Many different chains of different lengthsEach length ends with a labelled nucleotideMixture of lengths of DNA separated using electrophoresisThe shorter the length of DNA the faster it travelsComputer records the colours as they pass the end of the tube, if there are enough fragments then every base in the complete chain will be represented.Computer works out the sequence of the length of DNA
12Sequencing a genome Process is largely automated Put your DNA sample into a sequencing machineGet a print out from the bottomPreparation of DNA and analysis is still time consuming
13Learning OutcomesOutline how gene sequencing allows for genome-wide comparisons between individuals and between species.
14Comparing GenomesComparative gene mapping has a wide range of applicationsIdentification of genes for proteins gives clues to relative importance of these genes to lifeModelling the effects of changes to DNA can be carried outCompare pathogenic and non-pathogenic organismsIdentify targets for drug treatments and vaccinesAnalysis of individuals DNAPresence of alleles associated with diseaseDetermine evolutionary relationshipsClassification of organisms
15Learning Outcomes Define the term recombinant. Explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product.Describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes.
16Genetic Engineering Genetic engineering Recombinant DNA the use of technology to change the genetic material of an organism.Involves taking genes from an organism or one species and placing them in anotherRecombinant DNADNA that contains lengths of DNA from different speciesRecombinant organismOrganism to which the new gene has been addedAKA transgenic organism or transformed organism
17Gene transfer Identify gene that is required Cut out of chromosomesMade by “reverse transcription” of mRNAMultiple copies make using PCR (polymerase chain reaction)Gene inserted into a vectorVector is an organism or structure that can deliver the gene into required cells e.g. Plasmid, bacteriophage, liposomesVector inserts gene into cellsTransformed cells identified and cloned
18Extracting the geneA length of DNA known to contain HGH gene is treated with restriction enzymesRestriction enzymesCut DNA at specific base sequencesBamH1 always cuts DNA where there is a GGATCC sequence on one DNA strandCut the two DNA strands at different positions, leaving sticky endsShort lengths of unpaired bases on both pieces.
20Extracting the gene After cutting with restriction enzymes Get a mixture of lengths of DNARequired length of DNA can be identified usingDNA probesElectrophoresisMultiple copies of the DNA made using PCR.
21Learning OutcomesExplain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase.State other vectors into which fragments of DNA may be incorporated.
22Inserting gene into vector Plasmids are used if the gene is to be inserted into a bacteriaPlasmids often contain genes that confer resistance to antibiotics
23Inserting the HGH gene into plasmid Plasmid cut using the same restriction enzymeLeaves sticky ends that are complementary to those on the HGH genePlasmids and HGH genes are mixed togetherSticky ends of plasmid match up with sticky ends of HGH geneDNA ligase used to link the deoxyribose-phosphate backbonesProduce a closed circle of double stranded DNA containing HGH genenot all plasmids will take up HGH gene.
25Getting plasmids into bacteria Plasmids are mixed with a culture of bacteriaCalcium ions are added to affect the cell walls and plasma membranes1% of bacteria take up the plasmids containing the HGH gene
26Sorting out the transformed bacteria Plasmid pBR322 contains two antibiotic resistance genesTetracyclineAmpicillinThe restriction enzyme BamH1 cuts right through the tetracycline-resistance gene.So when HGH gene is inserted it inactivates the tetracycline resistance gene.
27Replica platingThe bacteria are grown on agar jelly containing ampicillinAny that survive have taken up the plasmidSamples of each colony are grown on a plate containing tetracyclineColonies that are unable to grow must have taken up the HGH geneThese colonies are selected from the first plate
28HGH productionThe genetically modified bacteria are cultured on a large scale in fermentersThey secrete HGHHGH is extracted, purified and sold
29VectorsThe method for getting the vector into the cell depends on the type of cellElectroporationHigh voltage pulse used to disrupt membraneMicroinjectionViral transferTi plasmidsLiposomes
30Learning OutcomesOutline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments.
31Polymerase Chain reaction Stage 1 The reactants are mixed together in a PCR vial.The mixture contains the DNA which is to be amplified, the enzyme DNA polymerase, small primer sequences of DNA and a good supply of the four nucleotide bases A,T,C and G.The vial is placed in a PCR machine.
32Polymerase Chain reaction Stage 2 The reaction mixture is heated to 90-95oC for about thirty seconds.At this temperature the DNA strands separate as the hydrogen bonds holding them together break down.
33Polymerase Chain reaction Stage 3 The mixture is cooled down to 55-60oC. At this temperature the primers bind (or anneal) to the single DNA strands.The primers are short sequences of nucleotide bases which must join to the beginning of the separated DNA strands for the full copying process to start.
34Polymerase Chain reaction Stage 4 In the final step the mixture is heated up again to 75oC for at least a minute.This is the optimum temperature for the DNA polymerase enzyme.The enzyme adds bases to the primers segments to build up complementary strands of DNA identical to the original molecule.
35PCRThese last three steps can be repeated around thirty times to give around 1 billion copies of the original DNA.The whole process takes only about 3 hours – and much of that is the time taken heating and cooling the reaction mixture in the PCR machine
36Summary of PCRDenaturing of double-stranded DNA molecules to make single strandedHigh temperature 95oCAnnealing primers to the ends of the single-stranded DNA molecules55-60oCBuilding complete new DNA strands using DNA polymerase72oC
40Learning OutcomesOutline how DNA fragments can be separated by size using electrophoresis.Describe how DNA probes can be used to identify fragments containing specific sequences.
41ElectrophoresisElectrophoresis separates different fragments of DNA according to their sizes.Tank set up containing agarose gelDirect current is passed continuously through the gelDNA fragments carry a small negative electric chargeDNA fragments are pulled through the gel towards the anodeThe smaller the fragments the faster they move through the agarose matrix.
42When the current is turned off DNA fragments will have ended up in different placesThese can be transferred onto absorbent paper or by a technique called southern blotting
44A radioactive probe is added to bind to the invisible bands of DNA, so they can blacken an X-ray film
45electrophoresisAfter electrophoresis and labelling of DNA samples, you can compare the DNA from different individuals.This is DNA profiling
46Gene ProbesA gene probe is a length of single stranded DNA that has a complementary base sequence to the gene you want to extractThe probe is “labelled”E.g. with nucleotides containing an isotope of phosphorous, 32P, which emits beta radiationWhen the probe is mixed with DNA fragments it forms hydrogen bonds with stretches of DNA complementary to its own base sequence (annealing)
47Using probes Probes can be used to locate specific sequences Identify the same gene on a variety of different genomesLocate a specific desired geneIdentify the presence or absence of an allele for a genetic disease
49Automated DNA Sequencing Previous methods of DNA sequencing were slow and time consuming.The current cutting edge approach uses an automated process involving interrupted PCR with modified nucleotide bases.
50The PCR sequence starts as before, with the primer annealing to the DNA fragment, allowing the DNA polymerase to attach.The DNA polymerase starts to add complementary nucleotides.Eventually, a modified nucleotide will be added, which prevents addition of any further nucleotides to the DNA strand.This generates many fragments of DNA that all end in a modified nucleotide, located in different positions on the unknown strand.These fragments are read by the automated sequencer, and the unknown sequence is revealed.
51The PCR mixture contains: PrimersDNA polymeraseSurplus nucleotide basesMultiple copies of the single stranded DNA fragment to be sequencedModified nucleotides with different coloured fluorescent markers
52An unknown sequence has a known initial fragment (CATGATA) Primer binds and free & tagged nucleotide bases are added with a polymerase enzyme.Terminator bases produce fragments of varying length.
53Electrophoresis allows fragments to be sorted by size, slowly revealing the complementary sequence to the unknown section.The sequence of fluorescent colours is then read by a laser, providing the complete sequence of bases.
54Learning OutcomesExplain how plasmids may be taken up by bacterial cells in order to produce a transgenic micro organism that can express a desired gene product.Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment.Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid.
55Genetic markersTo identify transformed bacteria the following genetic markers can be usedAntibiotic resistance genesGene that causes fluorescencefluoresces bright green in UV light
56Learning OutcomesOutline the process involved in the genetic engineering of bacteria to produce human insulin.
57Human Insulin production Stages to isolate the geneRemove mRNA from β-cells in islets of langerhansIncubate mRNA with reverse transcriptaseProduces complementary single stranded DNAThis is converted to double stranded DNA – insulin gene
58Human Insulin production Preparing the gene and vectorAdd lengths of single stranded DNA made from guanine nucleotides to create “sticky ends”Lengths of cytosine nucleotides were added to the cut ends of the plasmids
59Learning OutcomesOutline the process involved in the genetic engineering of Golden RiceTM.
60Golden Rice TM Vitamin A Required for the formation of rhodopsin Involved in the synthesis of glycoproteinsNeeded for the maintainance and differentiation of epithelial tissues and helps to reduce infectionEssential for bone growth
61Sources of Vitamin A in the diet Meat products, esp. Liverβ-carotene (precursor) in carrots – can be used to make retinolIn countries where vitamin A deficiency is significant they rely on rice as there staple food.
62Golden RiceTM Two genes were inserted into the rice genome Gene coding for phytoene synthase (daffodils)Gene coding for carotene desaturase (bacterium Erwinia uredovoraThe first rice produced did not produce significant quantities of β-carotene
64Golden RiceTMVersions were made of Golden RiceTM using genes from the maize plant and the rice itself.
65Are GMOs safe? Do they pose risks to health? Do they damage the environment?Are they hugely beneficial to humans and the environment?Two issuesCould genetically modified crops cause harm to other organisms in the environment?Is it safe to eat food from genetically modified plants?
66GM cropsThe majority of GM crops have been developed to benefit the grower and the retailer.Would GM crops be more acceptable if the benefits to health were clearly demonstrated?
67Learning OutcomesOutline how animals can be genetically engineered for xenotransplantation.
68xenotransplantationTransplanting tissues or organs between animals of different species.Human organ transplantationShortage of organsRejection of transplanted tissueCompatability checkedImmunosupressor drugs
69xenotransplantation Using organs from a pig Similar size and structure to human organsRisk of human immune responseHuman antibodies attach to glycoproteins on pig plasma membranesOne of these glycoproteins is made by an enzyme GGTA1 (1, 3-galactosyltransferase)If the sugar is not present, then antibodies don’t attach and immune attack is weakened.Genetically engineered pigs do not contain the gene that codes for the GGTA1 enzyme.
70Physiological problems Slight differences in organ sizeIs knocking out one gene enough to reduce the immune response sufficientlyBody temperature of pigs is 39oCPigs have much shorter lifespans than humans
71Ethical and medical problems Is it right to genetically modify pigs for our benefit?Is it acceptable to place an organ from another animal into a human body?Religious beliefsDisease transfer from pigs to humans
72Learning Outcomes Explain the term gene therapy. Explain the differences between somatic cell gene therapy and germ line cell gene therapy.
73Gene TherapyGene therapy is the treatment of a disease by manipulating the genes in a person’s cells.Two examples of gene therapySCIDCystic fibrosis
74SCID SCID severe combined immunodeficiency disease Caused by a faulty allele coding for the enzyme adenosine deaminase (ADA)This enzyme is essential for the healthy working of the immune system
75Gene therapy for SCID Gene therapy Alternative treatment Removal of patient’s T cells and insertion of the correct allele into them using a vector (retrovirus)Cells that have taken the allele up successfully are cloned and replaced into the patient’s bodyAlternative treatmentDaily injections of adenosine deaminase
76Problems with SCID gene therapy Some patients who appeared to have been successfully treated, went on to develop leukaemiaIs the risk of cancer acceptable when the patients would have died anyway from this rare and fatal disease?
77Cystic FibrosisAbnormally thick mucus is produced in the lungs and other parts of the body.Caused by a recessive allele of the gene that codes for the CFTR protein.CFTR geneSits on chromosome 9Commonest defective allele is a result of the deletion of three basesMachinery of cell recognises that the protein is not right and does not insert it in the cell membrane
78The CFTR protein forms channels for chloride ions in the plasma membrane
79Gene therapy for cystic fibrosis Normal allele inserted into liposomesSprayed as an aerosol into the noseLiposomes are lipid soluble and able to move through the lipid layers of the plasma membrane of the cells lining the respiratory passagesEffect only lasted a weekCells have a short lifespan and are continually replacedIntroducing the gene using adenovirusUnpleasant side effects – trials stopped
80Gene Therapy Somatic therapy Germline therapy Body cells genetically modifiedModified genes will not be passed on to any offspringGermline therapyChanging genes in cells that would go on to form gametesAll of the cells in the new organism would carry the genetic modificationModified genes in gametes could be passed on to offspring
82Germline cells Each cell of an early embryo is a stem cell It can divide and specialise to become any cell type within the bodyIt has the potential to become a new beingThese are germline cells
83Learning OutcomesDiscuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms.
84Benefits and risks of genetic engineering organismbenefitRiskMicro-organismGM bacteria can be used to produce useful productsAntibiotic resistance genes are used as genetic markersPlantsAnimalsHumansUse your textbooks to complete this table.