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Flashcards

Recombinant DNA technology

megan trent A level Biology Inglês 107 flashcards

sequencing projects have read entire genomes

Flashcards do conjunto (107)

  1. 1Using genome projectsSequencing projects have read the genomes of a wide range of organisms, including humans.
    Determining the genome of simpler organisms allows the sequences of the proteins that derive from the genetic code (the proteome) of the organism to be determined.
    This may have many applications, including the identification of potential antigens for use in vaccine production.
    In more complex organisms, the presence of non-coding DNA and of regulatory genes means that knowledge of the genome cannot easily be translated into the proteome.
    Sequencing methods are continuously updated and have become automated.
  2. 2Information from sequencing different organisms may provide information to helpcure disease, or allow us to use genes that provide specific adaptations in some organisms such as withstanding extreme or toxic environments, cleaning up pollutants or manufacturing biofuels.
  3. 3The sequencing of the human genome would not have been possible without the use ofbioinformatics
  4. 4What is bioinformatics?Bioinformatics is the branch of biology that is concerned with the acquisition, storage, and analysis of the information found in nucleic acid and protein sequence data. Computers and bioinformatics software are the tools of the trade
  5. 5Sequencing genomesWhole-genome shotgun sequencing involves cutting DNA into many small, easily sequenced sections and using computer algorithms to align overlapping segments to assemble the entire genome
  6. 6sequencing projects have read entire genomessequencing projects have read entire genomes
  7. 7sequencing the genome of simple organisms helps identify their proteinssequencing the genome of simple organisms helps identify their proteins
  8. 8its harder to translate the genome of complex organismsits harder to translate the genome of complex organisms
  9. 9sequencing methods are continuously updatedsequencing methods are continuously updated
  10. 10Medical advances as a result of the human genome projectSingle nucleotide polymorphisms (SNPs) have been found in the human genome.
    SNPs are single base variations in the genome (caused by a point mutation).
    They can be associated be disease and other disorders.
    Medical screening allows identification of potential medical problems and allows for early intervention to treat them
  11. 11The proteomeAll the proteins produced by a given type of cell (cellular proteome) at a given time, under specified conditions.
    All the proteins produced by the genome (complete proteome)
  12. 12Determining the proteome of simple organismsRelatively easy
    Prokaryotes have one, circular piece of DNA not associated with histones
    No non-coding portions of DNA
  13. 13human genome project ApplicationsIdentification of the proteins on pathogens that act as antigens to develop vaccines
    Classification of bacteria
    Understanding metabolism of bacteria
  14. 14Determining the genome and proteome of complex organismsMore difficult to do as many non-coding genes and regulatory genes
    In humans only 1.5% of genes may code for proteins
  15. 15DNA probesShort, single stranded length of DNA with a label attached
    Eg radioactively labelled probes
    Fluorescently labelled probes
  16. 16radioactively labelled probes –nucleotide sequence with the isotope 32P. The probe can be identified using x-ray film that is exposed to radioactivity
  17. 17Fluorescently labelled probes –emit light under certain conditions i.e. when the probe has bound to the target DNA sequence
  18. 18DNA probes detect specific sequencesDNA probes detect specific sequences
  19. 19DNA Probes Base sequences complementary tothe allele of the gene we want to find
  20. 20DNA Probes The double stranded DNA to be tested hasits strands separated by heating the DNA.
  21. 21DNA Probes The separated strands arecooled and mixed with the probe
  22. 22DNA Probes If the probe is complementary to a section of the DNA strand itwill bind. This is DNA hybridisation
  23. 23DNA Probes The site where the probe binds can beidentified by the radioactivity or fluorescent label
  24. 24DNA probes DNA hybridisationDNA probes DNA hybridisation
  25. 25Locating specific alleles of genes1. The sequence of nucleotides on the mutated gene is determined by DNA sequencing (Genetic libraries store DNA sequences responsible for many common genetic diseases)
    2. Fragment of DNA with complementary bases to mutant allele of gene produced
    3. DNA probe is formed by fluorescently labelling DNA fragment
    4. PCR techniques used to produce multiple copies of DNA probe
  26. 26Locating specific alleles of genesDouble stranded DNA denatured by heating
    Probe added to single stranded DNA fragments from person being screened and strands cooled
    If donor has mutated sequence the DNA probe will bind to the complementary sequence on the donor DNA
    DNA washed to remove unattached probes
  27. 27Locating specific alleles of genesThe presence of bound DNA probe is detected by shining light on the fragments causing the probe to fluoresce which can be seen using a special microscope
  28. 28It is possible to fix hundreds of probes in an array on a glass slide This means whendonor DNA is added to the slide many different genetic disorders can be tested simultaneously
  29. 29Uses of genetic testingCarrier screening which identifies unaffected parents who carry a recessive allele (heterozygous e.g. Cc) for an inherited disease, that requires homozygous recessive genotype (cc) to be expressed e.g. cystic fibrosis
  30. 30Uses of genetic testingpre-implantation genetic diagnosis
    pre-natal diagnostic testing
    newborn baby screening
    pre-symptomatic testing for predicting adult-onset disorders such as Huntington's disease
    pre-symptomatic testing for estimating the risk of developing adult-onset cancers eg mutations in tumour suppressor genes
    Confirmation that an individual has a suspected disease
    forensic/identity testing
  31. 31Personalised medicinePersonalised medicine
  32. 32Examples of using a personalised medicine approachPrescribing painkillers – many pain medications need a specific enzyme to activate them.
    About ½ the population have genes which alter the function of this enzyme. Screening will allow dosage of the painkiller to be adjusted to compensate for the genes effect on the metabolism of the drug to ensure the safe and effective use.
  33. 33Examples of using a personalised medicine approachVitamin E has been shown to reduce the risk of cardiovascular disease for people with diabetics and a certain genotype but will increase the risk for those with a different genotype.
    Screening will ensure the correct advice is given.
  34. 34Advantages and Limitations of gene testingAdvantages and Limitations of gene testing
  35. 35Many in the medical establishment feel that uncertainties surroundingtest interpretation, the current lack of available medical options for these diseases, the tests' potential for provoking anxiety, and risks for discrimination and social stigmatization could outweigh the benefits of testing.
  36. 36Ethical issues of genomic knowledge It is now possible to carry out automated screening to identify mutated genes in a sample of DNA This presents serious issues:Who owns the genetic information?
    Who has access to the genetic information?
    Insurers may discriminate against those with genes for say Alzheimer’s
    Knowledge of genome could lead to social stigmatization.
    Can parents test their children for adult-onset diseases?
    Couples will need to decide if they want a child with a genetic defect
    Could be used to choose or modify characteristics of a child
  37. 37Genetic counselling is the process of:evaluating family history and medical records
    Explaining the probability of passing on specific alleles
    Explaining the emotional, psychological, medical, social and economics
    consequences of genetic diseases to help with family planning
    ordering genetic tests
    evaluating the results of this investigation
    Patients screened at regular intervals
    Advice on lifestyle changes
    helping parents understand and reach decisions about what to do next
  38. 38Genetic counsellingHelping people faced with a diagnosis of genetic disease to understand both the factual information about the disease and the effect it will have on their lives, so that they can reach their own decisions about the future.
    Or help people with genetic disease decide whether to plan for a baby.
  39. 39Recombinant DNA technologyRecombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
    Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism.
    Fragments of DNA can be produced by several methods, including:
    • conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
    • using restriction enzymes to cut a fragment containing the desired gene from DNA
    • creating the gene in a ‘gene machine’. Fragments of DNA can be amplified by in vitro and in vivo techniques.
  40. 40Recombinant DNA technology The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments. The culture of transformed host cells as an in vivo method to amplify DNA fragments.• The addition of promoter and terminator regions to the fragments of DNA.
    • The use of restriction endonucleases and ligases to insert fragments of DNA into vectors. Transformation of host cells using these vectors.
    • The use of marker genes to detect genetically modified (GM) cells or organisms.
    (Students will not be required to recall specific marker genes in a written paper.)
  41. 41Genetic engineering UsesTransfer genes into bacteria so they can make useful products e.g. Insulin
    Transfers genes into plants and animals enabling them to acquire new characteristics e.g disease resistance
    Gene therapy
  42. 42Recombinant DNA Technology stages1. Isolation of the DNA fragments containing the gene for the desired protein
    2. Insertion of the DNA fragment into a vector
    3. Transformation – Transfer of DNA into a suitable host cell
    4. Identification – finding those host organisms containing the vector and DNA (by use of gene markers)
    5.Growth/Cloning – of the successful host cells
  43. 43DNA identification and isolationBefore a gene can be transplanted, it must be identified and isolated from the rest of the DNA. There are several methods of producing DNA fragments
    1.Conversion of mRNA to cDNA using reverse transcriptase
    2. Using restriction endonucleases to cut fragmetns containing the desired gene from DNA
    3.Creating the gene in a gene machine, usulaly based on a known protein structure
  44. 44Reverse TranscriptaseFinding correct piece of DNA containing the desired gene is difficult and cell only has 2 strands of DNA.
    In cytoplasm of cells large numbers of mRNA molecules.
    In pancreatic cells the functional mRNA for insulin in large quantities in cytoplasm.
    This mRNA can be extracted.
    A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase.
  45. 45Reverse Transcriptase diagramReverse Transcriptase diagram
  46. 46using reverse transcriptaseusing reverse transcriptase
  47. 47Using reverse transcriptase to make the human gene for insulin productionUsing reverse transcriptase to make the human gene for insulin production
  48. 48Restriction EndonucleasesRestriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict” viral growth)
    Restriction endonucleases cut up viral DNA
    There are many different types of restriction endonucleases. Each one cuts a DNA double strand at a specific sequence of bases called a recognition site.
    This property can be useful in gene technology to cut DNA at precise places ("molecular scissors").
    Short lengths of DNA cut out by restriction enzymes are called restriction fragments.
  49. 49Restriction endonucleases They cutphosphodiester bonds in the middle of the polynucleotide chain.
    Have highly specific active sites.
    Cut DNA at specific sites – about 4-8 base pairs long – these are called recognition sequences.
    This cut can be between two opposite base pairs. This leaves two straight edges known as blunt ends.
  50. 50Restriction Endonucleases – “Blunt Ends”Restriction Endonucleases – “Blunt Ends”
  51. 51Restriction Endonucleases – “Sticky Ends”Some restriction endonucleases cut DNA in a staggered fashion. This leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases.
    These recognition sites are usually palindronic, which means the sequence and its complement are the same but reversed.
    E.g. GAATTC and the complement CTTAAG
  52. 52Restriction Enzymes – “Sticky Ends”Restriction Enzymes – “Sticky Ends”
  53. 53using restriction endonucleaseusing restriction endonuclease
  54. 54The ‘gene machine’ It is now possible to manufacture genes in a laboratory.1. The amino acid sequence of a desired protein is determined
    2. The mRNA sequence can then be worked out by looking up the mRNA codons and the DNA triplets are worked out
    3. This sequence is fed into a computer
    4. sequence is checked for biosafety and biosecurity to ensure it meets international standards
  55. 55The ‘gene machine’ continued5. The computer designs a series of small, overlapping strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene
    6. In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence
    7. The oligonucleotides are joined together to make a gene containing only exons
    8. The gene is replicated using PCR (polymerase chain reaction)
  56. 56The ‘gene machine’ continued9. The complementary strand is synthesised using PCR to make a double stranded gene. This is then multiplied
    10. Using sticky ends the gene can be inserted into a bacterial plasmid.
    11. The genes are checked using standard sequencing techniques and those with errors are rejected
    These artificial genes can be produced in a short period of time and only contain exons so can be transcribed and translated by prokaryotic cells
  57. 57using a gene machine overviewusing a gene machine overview
  58. 58In vivo gene cloning – the use of vectorsOnce DNA has been cut into fragments it is necessary to find the fragment which has the required gene amongst the rest
    DNA probes can be used to identify these
    The fragments now need to be cloned by:
    In vivo by transferring the fragments to a host cell using a vector
    In vitro, using the polymerase chain reaction
  59. 59In vivo gene cloning step 1In vivo gene cloning step 1
  60. 60In vivo gene cloning step 2In vivo gene cloning step 2
  61. 61In vivo gene cloning step 3In vivo gene cloning step 3
  62. 62In vivo gene cloning to produce proteins you need promotor and terminator regionsIn vivo gene cloning to produce proteins you need promotor and terminator regions
  63. 63Insertion of DNA into a vector used to transport DNA into a host cell.VECTOR
  64. 64Insertion of DNA into a vector the most commonly used vector?PLASMID – the most commonly used vector. A circular piece of DNA found in bacteria.
    Plasmids are useful because the nearly always contain antibiotic resistance genes (see later).
  65. 65Insertion of gene into a Vector diagramInsertion of gene into a Vector diagram
  66. 66Importance of “sticky ends”DNA from different species can be joined together IF they have the same sticky ends – the same recognition site.
    In order to have the same sticky ends they must have been cut using the same restriction endonuclease.
    Sticky ends are joined together using DNA ligase to join the sugar-phosphate backbone together.
    The new DNA molecule is called recombinant DNA.
  67. 67Preparing the DNA fragment for insertionExtra lengths of DNA are added to the fragment
    These lengths of DNA contain a promoter sequence which is the binding site for RNA polymerase
    The nucleotide sequence of the promoter attach both RNA polymerase and transcription factors
    If we want the DNA fragment to be transcribed and then translated into a protein it is essential to attach the promoter region
    A length of DNA is also added at the end of the gene
    This is the terminator sequence which releases RNA polymerase and ends transcription.
  68. 68Introduction of DNA into host cells – TransformationThe plasmids must be reintroduced into the host cell e.g. bacteria
    This process is called transformation.
    The bacteria, plasmids and calcium ions are mixed together.
    By altering the temperature (and including calcium ions ion the mixture) the bacterial membranes become permeable and the plasmid can pass through the cell-surface membrane into the cytoplasm.
  69. 69Identification (step 4) of bacteria containing the plasmidOnly about 1% of bacterial cells take up any DNA/Plasmids when the two are mixed together.
    Firstly, we must identify the bacteria containing the plasmids – we do this by growing the bacteria on a medium containing an antibiotic.
    The antibiotic resistant gene is found in the plasmid only and therefore the bacteria that survive contain must contain the plasmid.
  70. 70Identification (stage 4) of bacteria containing the plasmid with the DNA fragmentGene markers are used to identify which plasmids have taken up the DNA fragment.
    Gene markers can be:
    - Resistance to an antibiotic
    - A fluorescent protein
    - An enzyme whose action can be identified
    Usually the gene marker is disrupted if the DNA fragment is present.
  71. 71The PlasmidThe ampicillin resistance gene is disrupted when the restriction enzymes cuts open the plasmid.
  72. 72Antibiotic-resistance MarkersThe second antibiotic-resistance gene (e.g. resistance to ampicillin) is used to identify those plasmids with a DNA fragment in them.
    If the DNA fragment has been inserted into the ampicillin resistance gene it will no longer grow on medium containing ampicillin.
    In order to identify these bacteria we use a process called replica plating.
  73. 73Replica PlatingThe bacteria are grown on standard nutrient agar, so all bacteria cells grow to form colonies.
    Some cells from the colonies are transferred onto agar that has been made with ampicillin, so that only those that have taken the plasmid will grow.
    Some cells from these colonies are transferred onto agar that has been made with tetracycline so only those that have taken up a plasmid that does not have the insulin gene will grow.
  74. 74Replica PlatingBy keeping track of which colonies are which, we know that any bacteria that grow on the ampicillin agar, but not on the tetracycline agar, must have taken up the plasmid with the insulin gene.
    We can now identify the colonies we want and grow them on a large scale. These bacteria then produce insulin on a large scale in a Fermentor which can be harvested for use.
  75. 75Inserting gene into a plasmid Enzyme markerInserting gene into a plasmid Enzyme marker
  76. 76Inserting gene into a plasmid Enzyme markerInserting gene into a plasmid Enzyme marker
  77. 77Inserting gene into a plasmid Enzyme markerInserting gene into a plasmid Enzyme marker
  78. 78Inserting gene into a plasmid Enzyme markerInserting gene into a plasmid Enzyme marker
  79. 79Inserting gene into a plasmid Enzyme markerInserting gene into a plasmid Enzyme marker
  80. 80Fluorescent markersA more recent marker gene
    Jellyfish have a gene which codes for a green fluorescent protein (GFP)
    The gene to be cloned is inserted in the middle of the GFP gene
    Any bacteria which takes up the plasmid with the gene in will not produce GFP and will not fluoresce
    Cells which have not taken up the gene will fluoresce
    No need for replica plating just select the colonies which do not fluoresce.
  81. 81Cloning (stage 5) the bacteriaFollowing successful identification of the bacteria containing the plasmid AND the DNA fragment, the bacteria are cloned in suitable containers with suitable conditions. (fermenter)
    As the bacteria are cloned, so is the plasmid containing the DNA fragment.
    This type of gene cloning is in vivo (cloned within a living organism).
  82. 82Donor DNA –a gene that is isolated for insertion.
  83. 83Plasmidscircular loops of DNA found in bacteria. The plasmid is known as a vector.
  84. 84Restriction enzymes –enzymes which cut DNA molecules between specific base sequences.
  85. 85DNA ligases –enzymes which join together portions of DNA.
  86. 86Sticky ends –the two ends of the ‘foreign’ DNA segment. They have a short row of unpaired bases that match the complementary bases at the two ends of the opened-up plasmid.
  87. 87Recombinant DNA –DNA which results from the combination of fragments from two different organisms.
  88. 88Reverse transcriptases –enzymes used to synthesize DNA from mRNA in specific cells.
  89. 89Clone –a population of genetically identical cells or organisms.
  90. 90Hazards of Recombinant DNA Technologybacteria readily exchange genetic material.
    deliberate use of antibiotic resistant genes in E. coli which lives in the human gut means that these genes could be accidentally transferred to human pathogens
    The possibility of transfer of DNA with linked pathogenic genes, for example, oncogenes thus increasing cancer risks
  91. 91PCR – polymerase chain reaction Objectives –1. Describe how PCR is used to amplify a section of DNA
    2. Describe the role of PCR in genetic fingerprinting in solving a crime or paternity
  92. 92In vitoIn vito
  93. 93In vito continuedIn vito continued
  94. 94compare in vitro gene cloning and in vivo gene cloningcompare in vitro gene cloning and in vivo gene cloning
  95. 95Evaluation of DNA Technology Microorganisms can be modified to producea wide range of substances
    Eg – antibiotics, hormones (insulin), enzymes
  96. 96Evaluation of DNA Technology Microorganisms can be used tocontrol pollution eg Oil and harmful gases
  97. 97PharmingGenetically modified plants can be transformed to produce a specific substance in a particular organ of the plant. These organs can then be harvested and the desired substance extracted. If a drug is involved this is called plant pharming.
    Produce plants that could manufacture antibodies or antigens
  98. 98transformed organisms are made using recombinant DNA technologytransformed organisms are made using recombinant DNA technology
  99. 99recombinant DNA technology can be used to benefit humans 1. agriculturerecombinant DNA technology can be used to benefit humans 1. agriculture
  100. 100recombinant DNA technology can be used to benefit humans 2.recombinant DNA technology can be used to benefit humans 2.
  101. 101recombinant DNA technology can be used to benefit humans 3.recombinant DNA technology can be used to benefit humans 3.
  102. 102there are concerns about the use of recombinant DNA technology agriculturethere are concerns about the use of recombinant DNA technology agriculture
  103. 103there are concerns about the use of recombinant DNA technology industrythere are concerns about the use of recombinant DNA technology industry
  104. 104there are concerns about the use of recombinant DNA technology medicinethere are concerns about the use of recombinant DNA technology medicine
  105. 105recombinant DNA technology also creates ownership. Here hows some parents:recombinant DNA technology also creates ownership. Here hows some parents:
  106. 106But humanitarians think it will benefit peopleBut humanitarians think it will benefit people
  107. 107—

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A level Biology FlashCards sobre Recombinant DNA technology, criado por megan trent em 03-04-2019.
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