OCR biology - gene technology
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| What are the uses of gene sequencing? | DNA profiling/ genetic fingerprinting (m/paternity testing, forensics), comparative genome mapping (importance, evolutionary relationships, disease causing genes -> targeted drug therapy, presence of alleles), genetic engineering, gene therapy
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| What is the function of electrophoresis? | Separate DNA fragments according to their size and arrange them into size order for further analysis
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| Briefly describe the method for gel electrophoresis | Treat DNA with restriction endonuclease enyzmes, cover plate with agarose gel, comb, cathode/anode/power supply, micropipette: sample + DNA size standard, immerse in buffer solution, pass electric current for 2 hours
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| Why do DNA fragments move + by how much? | DNA contains negatively charged phosphate groups, repelled by cathode, pass through agarose towards positive anode. Smaller fragments pass through agarose gel more quickly + easily so travel further in a fixed period of time
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| How can you analyse the agarose plate once completed? | Stain with ethidium bromide dye, place under UV, locate DNA fragments + compare to DNA size standard. Southern blotting: nylon/nitrocellulose sheet, paper towels, leave over night, heat gently, place under UV/ photographic film to locate markers/ probes
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| What is the function of the DNA size standard solution? | Contains DNA fragments of known length, use as a reference. Stain with ethidium bromide dye, UV light, compare distances travelled by size standard + unknown sample
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| What is the function of PCR? | Amplifies short DNA sequences, artificial DNA replication
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| Describe how PCR works | Mix DNA sample with free DNA nucleotides, polymerase. Heat to 92˚c for 20s to break H bonds, cool to 55˚c, add primers, anneal to 3' end by CBP, heat to 72˚c, polymerase attaches, extends primers, phosphodiester, 5'->3',amount increases exponentially
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| What is special about the DNA polymerase enzyme used? | Thermophilic, not denatured by high temperatures used in the process, derived from bacterium Thermus Aquaticus in hot springs
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| What is the difference between natural DNA replication and PCR? | Artificial uses primers instead of primase enzyme, cycle of heating/ cooling to separate/form H bonds instead of helicase, only replicates short DNA sequences
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| Describe the process of automated Sanger sequencing | Force DNA through tiny hoels under high pressure, gel electrophoresis, amplify each fragment length idnvidiaully by PCR, add dideoxynucleotides (terminator bases, tagged with fluorescent marker), if attached to growing DNA strand polyermase detaches..
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| Continued | Many copies of DNA sample terminate at different points with dideoxynucleotide at end, electrophoresis along capillary tube, smaller fragments reach anode faster, UV laser at anode, computer records sequence of colours -> bases, all bases are represented
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| Define: genetic engineering | Inserting a specific gene from one organism to another, producing a transgenic organism with recombinant DNA that expresses the new gene via protein syntehsis
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| Define: transgenic organism | Organism that has had DNA added to its cells via genetic engineering, now has recombinant DNA
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| Define recombinant DNA | DNA formed by joining sections of DNA from 2 sources together, often from 2 different species
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| Give 7 uses of genetic engineering | Xenotransplantation, gene therapy, animals with high milk/ meat yield, sheep that produce pharmaceutical chemicals in their milk, Golden Rice, plants with high yield/ resistance, E.coli that produces human insulin/ growth hormone
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| What is the 1st step for genetic engineering and how is it done? | Isolate gene for engineering: 1)locate desired gene with probe, restriction endonuclease enzymes cut out gene by hydrolysing sugar phosphate backbone at specific restriction sites, sticky ends anneal by cmp. 2)extract mRNA from cytoplasm of a cell...
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| Continued | That expresses the gene, reverse transcriptase synthesises a single strand of comeplemtayr cDNA, DNA polymerase forms double stranded DNA. 3)amino acid sequence --> base sequence, automated polynucleotide sequencer
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| What are sticky ends? | Short sequences of unpaired, exposed nucleotides at the end of a cut section of DNA that will anneal to each other by complementary base pairing, hydrogen bonding (also complementary to cut plasmid)
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| What is the 2nd step for genetic engineering and how is it done? | Insert gene into vector. Plasmids: lyse bacteria to extract plasmid, cut with same restriction endonuclease enzymes, mix gene/cut plasmids/DNA ligase, some plasmids are spliced, complementary sticky ends anneal by cmp, ligase seals recombinant plasmid
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| Continued | Virus DNA, yeast DNA, liposome
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| What is the 3rd step for genetic engineering and how is it done? | insert vector->recipient cell: bacteria (heat shock, Ca2+, transgenic), electroporation (high-voltage pulse), microinjection (inject DNA->nucleus), viral transfer (retroviruses), Ti plasmids (replace TDNA in agrobacterium tumefaciens), liposomes diffuse
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| What is the purpose of producing E.coli that can produce insulin? | Give insulin injections to people with type 1 diabetes MELLITUS
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| What is the 1st step for producing GM E.coli? | Isolate gene for human insulin: centrifuge pancreatic tissue (including islets of Langerhans), isolate mRNA of correct length, amplify by PCR, use reverse transcriptase to synthesise single stranded cDNA, DNA polymerase cDNA gene, sticky ends
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| What si the 2nd step? | Introduce cDNA gene to plasmids: lyse bacteria, cut open plasmids with restriction enzyme, mix cut plasmids/ cDNA gene/ ligase, some plasmids are spliced with cDNA gene (sticky ends anneal, DNA ligase seals sugar phosphate backbone), some reseal
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| What is the 3rd step? | Mix plasmids, bacteria, Ca2+ ions, heat shock, <1% of bacteria take up a plasmid, <0.25% take up a recombinant plasmid (become transformed, transgenic bacteria)
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| What is the 4th step? | Grow 3 types of bacteria (no plasmid, unchanged plasmid, recombinant plasmid) on nutrient agar, each cell forms a colony by binary fission
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| How do you identify the transformed bacteria? | GENETIC MARKERS. Replica plating: standard agar -> agar containing ampicillin (only bacteria with plasmid grow + reproduce) -> agar containing tetracycline (only non-transgenic bacteria grow+ reproduce), grow bacteria that survive on A not T in fermneter
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| How do you transfer bacteria between agar plates? | Block covered in sterile velvet
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| Define replica plating | Grow bacteria on standard nutrient agar plate then transfer to agar plates containing different growth promoters/ inhibitors, analysis of growth patterns reveals information about genetic properties of bacteria
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| What is conjugation? | Form of horizontal gene transfer: conjugation tube grows between donor and recipient, enzyme makes a nick in donor plasmid, plasmid replicates, strand not being used as a template moves into recipient, cells separate, plasmids close into loops
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| What is transformation? | Form of HGT: bacteria take up DNA (plasmids, transposons) from environment, released by the lysis of other bacteria
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| What is transduction? | Form of HGT: transfer of genes by infection with a bacteriophage
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| What are the advantages of HGT for bacteria? | Faster spread of genes conferring resistance to antibiotics, more genetic variation, transfer of a human gene to bacterium Neisseria Gonorrhoea allowed it to survive in humans
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| What is golden rice? | GM rice, biofortified, contains higher than normal concentration so beta-carotene, precursor for vitamin A, in the endosperm
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| How is golden rice produced? | 1) precursor -> phytoene - phytoene synthetase, gene from daffodil. 2) phytoene -> lycopene - Crt1 enzyme, gene from soil bacterium erwinia uredevora. 3) lycopene -> beta carotene - catalysed by enzymes in endosperm. Inserted near promoter sequence
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| What are the advantages of golden rice? | Treat vitamin A deficiency (causes blindness, common in areas of world where rice is a staple food), farmers are allowed to grow + replant seeds without paying a licence fee
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| What are the drawbacks of golden rice? | A large amount of rice would need to be eaten to treat vitamin A deficiency, genes transferred to wild relatives, less biodiversity, could be harmful in long term
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| What is gene therapy? | Treating disease by manipulating the genes in an individual's cells. A functioning copy of a gene is inserted into the cells so the functioning allele is transcribed instead of the faulty one -> correct polypeptide is translated
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| Why is it easier to treat recessive disorders? | A dominant allele can be added to the cells and have an effect: the mutant, recessive allele doesn't need to be removed
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| What is RNA interference? | siRNAs inserted into cell, binds to complementary mRNA produced by the transcription of a faulty gene by complementary base pairing/ hydrogen bonding, double-stranded RNA can't be translated so gene isn't expressed
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| How is gene therapy currently used? | Cancer therapy. RNA interference to block the expression of an oncogene. Make cancer cells more vulnerable to immune attack e.g. produce more cytokines, antigens
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| What is a probe? | Single-stranded piece of DNA, 50-80 nucleotides long, complementary to a section of the DNA being investigated, will anneal to this sequence by complementary base pairing/ hydrogen bonding. Radioactive marker - photographic film, fluorescent marker - UV
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| What are the uses of probes? | Locate gene for genetic engineering, comparative genome mapping, identify presence/ absence of mutant allele/ allele linked to disease
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| What is a primer? | Single-stranded piece of DNA, 10-20 nucleotides long, used in PCR, anneals to the 3' end of DNA template by complementary base pairing/ hydrogen bonding, double-stranded section of DNA for DNA polymerase to attach to
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| What is somatic cell gene therapy? | Modification of specific somatic cells, modification is limited to those cells only, don't pass on as specialised cells don't divide, doesn't pass to offspring
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| What is germline cell gene therapy? | Modification of embryos and gametes so every cell in an individual contains the modified gene, can be passed to offspeing
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| Compare somatic and germline cell gene therapy | S-somatic cells, G-gametes/ embryos. S-limited to certain cells, G-all cells in body. S-doesn't pass to offspring, G-does. S-difficult (ex vivo, vector), G-straightforward. S-fewer ethical issues, used for cancer/ cystic fibrosis, G-illegal, unethical
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| What is allotransplantation? | Transplantation of tissues/ organs bewteen animals of the same species
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| What is xenotransplantation? | Transplantation of tissues/ organs between animals of different species
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| How can animals be used in xenotransplantation? | Kill animals then harvest their organs, GM to make them suitable e.g. switch off GGAT1 gene in pigs that codes for glycoproteins on plasma membrane that result in rejection
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| What are the advantages of xenotransplantation? | Reduces waiting times on transplant list, GM to reduce chance of rejection e.g. GGAT1
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| What are the disadvantages of xenotransplantation? | Practical issues: differences in organ size, differences in lifespan e.g. pig organs would age prematurely, diseases may spread between species. Ethical issues: animal welfare issues, may be unsuitable for certain religious groups
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| What are the uses + drawbacks of genetic engineering in humans? | Use: treating disease. Drawbacks: people resulting from germline cell gene therapy have no say in the modification of their DNA, permanent, eugenics, designer babies, harmful/ unintended consequences
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| What are the uses + drawbacks of genetic engineering in animals? | Uses: xenotransplantation, sheep that produce pharmaceutical chemicals in their milk e.g. a anti-trypsin for hereditary emphysema, high meat/ milk yield. Drawbacks: practical issues, religious groups, animal welfare
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| What are the uses + drawbacks of genetic engineering in plants? | Uses: micropropagation, resistance to pests/herbicides/disease, golden rice. Drawbacks: less genetic variation as modified genes spread to wild relatives, genes for herbicide/pesticide resistance spread to weeds/pests, GM crops unsafe/ allergies
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| What are the uses + drawbacks of genetic engineering in microorganisms? | Uses: GM E.coli produces human growth hormone/ human insulin. Drawbacks: genes for antibiotic resistance are used as genetic markers -> increase in resistance, modified genes may spread to pathogens
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| After sequencing individual fragment of DNA with the Sanger process, how do you work out the entire sequence? | Map the sequenced DNA fragments to see where they have come from in the genome e.g. location of microsatellites. Repeat the process many times so DNA splits into different fragments, then identify overlapping sequences to reassemble whole genome
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