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F215 control, genomes and environment Module 2 – Biotechnology and gene technologies.

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1 F215 control, genomes and environment Module 2 – Biotechnology and gene technologies

2 Learning Outcomes  Outline the steps involved in sequencing the genome of an organism.

3 Genomes  1950’s  Learnt that DNA is the genetic material  Gene technology  Use of DNA to produce something that we want  Developing rapidly  Becoming more and more able to alter genes within organisms  Point to think about  Just because we can do something does that mean that we should do it?

4 Manipulating DNA  Advances in DNA technology  DNA profiling (genetic fingerprinting)  Genomic sequencing  Comparative genome mapping  Genetic engineering  Gene therapy

5 Genome  All 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.

6 Human Genome Project  1988  International project started to discover the sequence of bases in each of the 23 different types of chromosomes found in human cells  2000  A working draft sequence was produced

7 Facts about human genome  99.9% of the base sequence in our DNA seems to be identical in all humans  Variation is caused by the variable 0.1%  This 0.1% is very variable  Variations can be used for DNA profiling  2% of human genome codes for the manufacture of proteins  Giving around genes in the human genome (even mice have more!!)  The rest of the “junk” genome, may be involved in gene expression

8 Sequencing a genome  The genome is broken up and sequenced in sections  Sequencing is carried out on overlapping regions  Stages  Genome mapping  Mechanically break into smaller sections  Carry out sequencing on overlapping sections  Analyse and put back together to form the complete code

9 Sequencing a genome  Look at the worksheet  “sequencing a plant gene”  Make multiple copies of the genome using PCR  DNA randomly broken up into lengths 2000bp – 10000bp long  These lengths can then be broken up further

10 Sequencing a genome  Make multiple labelled copies of each small length of DNA  Lengths of DNA mixed with ▪ DNA Polymerase ▪ Primer ▪ “normal” DNA nucleotides ▪ “labelled” DNA nucleotides ▪ dideoxy nucleotides ▪ Four colours of dye used for bases A, T,G and C ▪ If incorporated in nucleotide chain – chain stops growing

11 Sequencing a genome  Result  Many different chains of different lengths  Each length ends with a labelled nucleotide  Mixture of lengths of DNA separated using electrophoresis  The shorter the length of DNA the faster it travels  Computer 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

12 Sequencing a genome  Process is largely automated  Put your DNA sample into a sequencing machine  Get a print out from the bottom  Preparation of DNA and analysis is still time consuming

13 Learning Outcomes  Outline how gene sequencing allows for genome-wide comparisons between individuals and between species.

14 Comparing Genomes  Comparative gene mapping has a wide range of applications  Identification of genes for proteins gives clues to relative importance of these genes to life  Modelling the effects of changes to DNA can be carried out  Compare pathogenic and non-pathogenic organisms ▪ Identify targets for drug treatments and vaccines  Analysis of individuals DNA ▪ Presence of alleles associated with disease  Determine evolutionary relationships  Classification of organisms

15 Learning 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.

16 Genetic Engineering  Genetic engineering  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 another  Recombinant DNA  DNA that contains lengths of DNA from different species  Recombinant organism  Organism to which the new gene has been added  AKA transgenic organism or transformed organism

17 Gene transfer  Identify gene that is required  Cut out of chromosomes  Made by “reverse transcription” of mRNA  Multiple copies make using PCR (polymerase chain reaction)  Gene inserted into a vector  Vector is an organism or structure that can deliver the gene into required cells e.g. Plasmid, bacteriophage, liposomes  Vector inserts gene into cells  Transformed cells identified and cloned

18 Extracting the gene  A length of DNA known to contain HGH gene is treated with restriction enzymes  Restriction enzymes  Cut DNA at specific base sequences  BamH1 always cuts DNA where there is a GGATCC sequence on one DNA strand  Cut the two DNA strands at different positions, leaving sticky ends ▪ Short lengths of unpaired bases on both pieces.

19 Cutting DNA with a restriction enzyme

20 Extracting the gene  After cutting with restriction enzymes  Get a mixture of lengths of DNA  Required length of DNA can be identified using ▪ DNA probes ▪ Electrophoresis  Multiple copies of the DNA made using PCR.

21 Learning Outcomes  Explain 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.

22 Inserting gene into vector  Plasmids are used if the gene is to be inserted into a bacteria  Plasmids often contain genes that confer resistance to antibiotics

23 Inserting the HGH gene into plasmid  Plasmid cut using the same restriction enzyme  Leaves sticky ends that are complementary to those on the HGH gene  Plasmids and HGH genes are mixed together  Sticky ends of plasmid match up with sticky ends of HGH gene  DNA ligase used to link the deoxyribose- phosphate backbones  Produce a closed circle of double stranded DNA containing HGH gene  not all plasmids will take up HGH gene.

24 Inserting the HGH gene into plasmid

25 Getting plasmids into bacteria  Plasmids are mixed with a culture of bacteria  Calcium ions are added to affect the cell walls and plasma membranes  1% of bacteria take up the plasmids containing the HGH gene

26 Sorting out the transformed bacteria  Plasmid pBR322 contains two antibiotic resistance genes  Tetracycline  Ampicillin  The restriction enzyme BamH1 cuts right through the tetracycline- resistance gene.  So when HGH gene is inserted it inactivates the tetracycline resistance gene.

27 Replica plating  The bacteria are grown on agar jelly containing ampicillin  Any that survive have taken up the plasmid  Samples of each colony are grown on a plate containing tetracycline  Colonies that are unable to grow must have taken up the HGH gene  These colonies are selected from the first plate

28 HGH production  The genetically modified bacteria are cultured on a large scale in fermenters  They secrete HGH  HGH is extracted, purified and sold

29 Vectors  The method for getting the vector into the cell depends on the type of cell  Electroporation ▪ High voltage pulse used to disrupt membrane  Microinjection  Viral transfer  Ti plasmids  Liposomes

30 Learning Outcomes  Outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments.

31 Polymerase 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.

32 Polymerase Chain reaction Stage 2  The reaction mixture is heated to o C for about thirty seconds.  At this temperature the DNA strands separate as the hydrogen bonds holding them together break down.

33 Polymerase Chain reaction Stage 3  The mixture is cooled down to o C. 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.

34 Polymerase Chain reaction Stage 4  In the final step the mixture is heated up again to 75 o C 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.

35 PCR  These 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

36 Summary of PCR  Denaturing of double-stranded DNA molecules to make single stranded  High temperature 95 o C  Annealing primers to the ends of the single-stranded DNA molecules  o C  Building complete new DNA strands using DNA polymerase  72 o C

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40 Learning Outcomes  Outline how DNA fragments can be separated by size using electrophoresis.  Describe how DNA probes can be used to identify fragments containing specific sequences.

41 Electrophoresis  Electrophoresis separates different fragments of DNA according to their sizes.  Tank set up containing agarose gel  Direct current is passed continuously through the gel  DNA fragments carry a small negative electric charge  DNA fragments are pulled through the gel towards the anode  The smaller the fragments the faster they move through the agarose matrix.

42  When the current is turned off  DNA fragments will have ended up in different places  These can be transferred onto absorbent paper or by a technique called southern blotting

43 Using electrophoresis

44  A radioactive probe is added to bind to the invisible bands of DNA, so they can blacken an X-ray film

45 electrophoresis  After electrophoresis and labelling of DNA samples, you can compare the DNA from different individuals.  This is DNA profiling

46 Gene Probes  A gene probe is a length of single stranded DNA that has a complementary base sequence to the gene you want to extract  The probe is “labelled”  E.g. with nucleotides containing an isotope of phosphorous, 32 P, which emits beta radiation  When the probe is mixed with DNA fragments it forms hydrogen bonds with stretches of DNA complementary to its own base sequence (annealing)

47 Using probes  Probes can be used to locate specific sequences  Identify the same gene on a variety of different genomes  Locate a specific desired gene  Identify the presence or absence of an allele for a genetic disease

48 Revision of DNA sequencing

49 Automated 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.

50  The 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.

51  The PCR mixture contains:  Primers  DNA polymerase  Surplus nucleotide bases  Multiple copies of the single stranded DNA fragment to be sequenced  Modified nucleotides with different coloured fluorescent markers

52 An 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.

53 Electrophoresis 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.

54 Learning Outcomes  Explain 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.

55 Genetic markers  To identify transformed bacteria the following genetic markers can be used  Antibiotic resistance genes  Gene that causes fluorescence ▪ fluoresces bright green in UV light

56 Learning Outcomes  Outline the process involved in the genetic engineering of bacteria to produce human insulin.

57 Human Insulin production  Stages to isolate the gene  Remove mRNA from β-cells in islets of langerhans  Incubate mRNA with reverse transcriptase ▪ Produces complementary single stranded DNA ▪ This is converted to double stranded DNA – insulin gene

58 Human Insulin production  Preparing the gene and vector  Add 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

59 Learning Outcomes  Outline the process involved in the genetic engineering of Golden RiceTM.

60 Golden Rice TM  Vitamin A  Required for the formation of rhodopsin  Involved in the synthesis of glycoproteins  Needed for the maintainance and differentiation of epithelial tissues and helps to reduce infection  Essential for bone growth

61 Sources of Vitamin A in the diet  Meat products, esp. Liver  β-carotene (precursor) in carrots – can be used to make retinol  In countries where vitamin A deficiency is significant they rely on rice as there staple food.

62 Golden Rice TM  Two genes were inserted into the rice genome  Gene coding for phytoene synthase (daffodils)  Gene coding for carotene desaturase (bacterium Erwinia uredovora  The first rice produced did not produce significant quantities of β- carotene

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64 Golden Rice TM  Versions were made of Golden Rice TM using genes from the maize plant and the rice itself.

65 Are GMOs safe?  Do they pose risks to health?  Do they damage the environment?  Are they hugely beneficial to humans and the environment?  Two issues  Could genetically modified crops cause harm to other organisms in the environment?  Is it safe to eat food from genetically modified plants?

66 GM crops  The 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?

67 Learning Outcomes  Outline how animals can be genetically engineered for xenotransplantation.

68 xenotransplantation  Transplanting tissues or organs between animals of different species.  Human organ transplantation  Shortage of organs  Rejection of transplanted tissue ▪ Compatability checked ▪ Immunosupressor drugs

69 xenotransplantation  Using organs from a pig  Similar size and structure to human organs  Risk of human immune response ▪ Human antibodies attach to glycoproteins on pig plasma membranes ▪ One 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.

70 Physiological problems  Slight differences in organ size  Is knocking out one gene enough to reduce the immune response sufficiently  Body temperature of pigs is 39 o C  Pigs have much shorter lifespans than humans

71 Ethical 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 beliefs  Disease transfer from pigs to humans

72 Learning Outcomes  Explain the term gene therapy.  Explain the differences between somatic cell gene therapy and germ line cell gene therapy.

73 Gene Therapy  Gene therapy is the treatment of a disease by manipulating the genes in a person’s cells.  Two examples of gene therapy  SCID  Cystic fibrosis

74 SCID  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

75 Gene therapy for SCID  Gene therapy  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 body  Alternative treatment  Daily injections of adenosine deaminase

76 Problems with SCID gene therapy  Some patients who appeared to have been successfully treated, went on to develop leukaemia  Is the risk of cancer acceptable when the patients would have died anyway from this rare and fatal disease?

77 Cystic Fibrosis  Abnormally 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 gene  Sits on chromosome 9  Commonest defective allele is a result of the deletion of three bases  Machinery of cell recognises that the protein is not right and does not insert it in the cell membrane

78 The CFTR protein forms channels for chloride ions in the plasma membrane

79 Gene therapy for cystic fibrosis  Normal allele inserted into liposomes  Sprayed as an aerosol into the nose  Liposomes are lipid soluble and able to move through the lipid layers of the plasma membrane of the cells lining the respiratory passages  Effect only lasted a week ▪ Cells have a short lifespan and are continually replaced  Introducing the gene using adenovirus  Unpleasant side effects – trials stopped

80 Gene Therapy  Somatic therapy  Body cells genetically modified  Modified genes will not be passed on to any offspring  Germline therapy  Changing genes in cells that would go on to form gametes  All of the cells in the new organism would carry the genetic modification  Modified genes in gametes could be passed on to offspring

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82 Germline cells  Each cell of an early embryo is a stem cell  It can divide and specialise to become any cell type within the body  It has the potential to become a new being  These are germline cells

83 Learning Outcomes  Discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms.

84 Benefits and risks of genetic engineering organismbenefitRisk Micro-organism GM bacteria can be used to produce useful products Antibiotic resistance genes are used as genetic markers Plants Animals Humans Use your textbooks to complete this table.


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