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54 Cards in this Set
- Front
- Back
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What are the uses of gene tech? (5) |
Repairing a genetic defect 'gene therapy';
enhancing an effect/increasing resistance to disease or damage (using transformed organisms); Medical diagnosis and treatment of disease; clinical screening and DNA PROBES; treatment of cancer; |
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What is a transgenic organism/genetically modified organism? |
One with recombinant DNA/DNA altered by insertion of genes from another organism |
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What is recombinant DNA? |
DNA mixed with that of another species/organism |
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What are the processes involved in producing a protein using DNA tech? |
Isolate of DNA fragment Insertion into a vector Transformation (inserting into suitable host cells) Identification (using gene markers) Cloning |
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What are the 2 ways of isolating a gene fragment? |
Reverse transcriptase restriction endonuclease |
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How can retroviruses be used to isolate a DNA fragment? |
Retroviruses have reverse transcriptase convert mRNA into DNA use enzyme to produce cDNA from mRNA DNA polymerase builds second strand using cDNA as template |
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Where do restriction endonucleases originate from? |
In bacteria as a defence mechanism Cuts viral DNA which viruses insert to take over the cell |
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How do restriction endonucleases function? |
Cut DNA at a specific recognition sequence EITHER cut in a staggered fashion at a palindromic sequence each strand has unpaired exposed bases 'sticky ends' as one is longer than other OR leaving blunt ends |
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What is a palindromic sequence? |
Sequence on one strand reads the same as the sequence on the other strand in the reverse direction |
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How can restriction endonucleases be used to isolate DNA fragments and insert them into a vector? |
Cut DNA at specific (palindromic) recognition site Cut in a staggered fashion, leaving 'sticky' ends (as one longer than other/exposed unpaired bases). Fragment can be joined with a vector using DNA ligase vector (plasmid or virus) has recombinant DNA |
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How are is DNA incorporated into a bacterium? |
DNA isolated and cut out using specific restriction endonuclease (cuts at recognition sites between gene) Same restriction endonuclease cuts plasmid/opens it up DNA ligase used to 'join' sticky ends together when mixed Recombinant plasmid taken up by bacterium using mixture of calcium ions and heat shock to increase permeability |
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Why won't all the bacterium (in in vivo cloning) posess a recombinant plasmid? |
Not all take up plasmid Some plasmids not recombinant/close up again/dont join with fragment |
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What are the different types of gene markers? |
Antibiotic resistance flourescent marker enzyme marker (produce lactase which turns substrate blue) |
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Why are gene markers required? |
Show which bacteria have taken up a recombinant plasmid |
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What is a flourescent marker gene? |
GFP produces green flourescent |
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What isa good plasmid for replica plating? |
R-plasmid, antibiotic resistance genes for ampicillin and tetracycline |
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How does replica plating work? |
Use plasmid with 2 resistance genes (R-plasmid) Cut open plasmid at one of the genes; combine plasmid with target gene using ligase use heatshock/calcium ions to transform bacteria; culture bacteria on plate with antibiotic 1; those without plasmid die, cant produce enzyme for resistance; transfer colonies onto second identical plate and treat with antibiotic 2 those that dont grow are good |
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What is the advantage of flourescent gene markers over antibiotic resistance? |
Don't have to kill bacteria/replica plating easy and rapid |
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What is added into a PCR machine?
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Thermostable DNA polymerase (taq) DNA fragment nucleotides primers (thermocycler) |
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Where does the taq polymerase come from? |
Thermophilic Thermus aquaticus |
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Describe PCR |
Heated to 95 degrees; separate strands of DNA cool to 55 degrees addition/annealing allow complementary base pairing of primers to ends of DNA fragments. primers provide starting sequence for polymerisation and prevent DNA strands from rejoining heat to 72 degrees - synthesis of DNA optimum temp for polymerase, starting at the primer adding complementary nucleotides |
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What is the function of the primer? |
Provide starting sequence from where DNA polymerase can join compelmentary nucleotides (as can only add to the end of existing chain) also prevents DNA strands from rejoining |
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What are primers? |
Short sequences of bases that are complementary to the end of a DNA fragment |
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Why do you need 2 diff primers in PCR? |
Because opposite ends of each strand have diff base sequences |
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What are the uses of PCR? |
Forensic analysis - tiny samples can be multiplied many times also genetic screening - enough to test for a particular disease |
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What are the advantages of PCR? |
It is very rapid and produces many copies little purification is needed/no living cells/no complex vulturing |
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What are the disadvantages of PCR? |
Mistakes very common + contaminants amplidfied |
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What are the advantages of in vivo cloning? |
1. Gene can be expressed and used to make large numbers of proteins for medical.commercial uses 2. Gene can be transferred into another organism easily (gene therapy) as already in vector 3. No risk of contamination as specific endonucleases 4. Mistakes not common as proof-reading enzymes/mutations are rare |
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What are the advantages of gene tech? |
Genes can be inserted into animals (produce hormones/enzymes/medicines or grow faster/disease resistant) genes can be inserted into micororganisms to produce antibiotics/hormones/enzymes on an industrial scale genes can be inserted into plants to improve crop yield/improve nutrition/disease resistant/pest resistant/herbicide resistant/produce medicine genetic fingerprinting/gene therapy/cloning/organs for transplant |
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What are the disadvantages of gene tech? |
May damage ecosystem through horizontal gnee transfer May have unknown long-term effects/cause cancer gene therapy may lead to designer babies cloning is a slippery slope genetic fingerprinting is easy to manipulate |
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What is gene therapy? |
The replacement of defective alleles with functional alleles (or supplementation - a copy of the dominant healthy allele is added alongside the defective allele) |
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What is cystic fibrosis and what are the symtoms? |
Recessive genetic disorder (CFTR) gene adenine-adenine-adenine dleeted, protein has one less amino acid, doesn't produce correct chloride ion channel protein no chloride ions transported out of epithelial cells, no osmosis out, mucus is thick infertility (mucus in sperm duct), congestion in lungs and infection, fibrous cysts due to blocked pancreatic ducts, breathing difficulties |
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What are the 2 different techniques of gene therapy? |
Germ-line gene therapy - replace or supplement allele in fertilised egg cell, all daughter cells have healthy allele somatic-cell gene therapy - targete affected tissues, but cells always die and get replaced, so repeated treatements (target stem cells so lifespan of one individual) |
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What are the steps involved in gene therapy? |
Isolate disease allele base sequence of healthy allele copy healthy allele insert/transform cells |
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What are the methods of delivering a healthy allele? |
Adenoviruses, liposomes |
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Describe how adenoviruses can be used to deliver a healthy allele? |
Made harmles sby interfering with replication grown in epithelial cells with plasmids with CFTR gnee incorporate healthy gene inhaled thorugh nostrils inject into epithelial cell |
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Describe how liposomes can be used to deliver a healthy allele? |
isolate healthy allele combine wiht plasmid replicate plasmid in host bacterial cell isolate and extract plasmid wrap plasmid in lipid molecule - liposome liposomes inhaled as aerosol and pass through phospholipid bilayer into epithelial cells |
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What are the issues with adenoviruses/liposomes? |
BOTH: effect is short lived, not passed onto daughter cells so repeat treatments needed/healthy allele not expressed ADENOVIRUS: may become harmful/may develop immunity LIPOSOME: might not pass through bronchioles |
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What are DNA probes? |
Short, single stranded pieces of DNA that have a radioactive or flourescent label attached used to identify the presence of a particular gene |
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How are DNA probes used to identify particular genes? |
DNA probe made complementary to sequence of gene sample of DNA is treated to separate strands strands mixed with probe, probe joins by DNA hybridisation Identify by fluorescence or radioactivity (using photographic film/UV light) |
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What are the 2 types of DNA probes? |
Radioactively labelled (using 32P) identified using photographic plate flourescently labelled identified using UV light |
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What is the alternative name for DNA sequencing? |
dideoxy sequencing |
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What is restriction mapping used for? |
Used to divide to gene into separate fragments to be sequenced ensured that order of each fragment in gnee is known |
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How does DNA sequencing work? |
4 diff testubes each with primers (labelled), polymerase, fragment, nucleoptides, and small quantity one of terminator (dideoxynucleotides) nucleotides terminator stops addition of more bases by polymerase enzyme as binding of dideoxy is random, due to number of fragments, a dideoxy will bind to every complementary site on DNA DNA fragments of diff sizes all fragments end with same base in a testtube |
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How does gel electrophoresis work? |
DNA sample placed on agar gel voltage applied, DNA fragments move to positive terminal Resistance of gel means larger framgments move slowly and less far over fixed period DNA bands made visible using UV light or photographic film |
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What is restriction mapping? |
Used to determine where restriciton sites are in a DNA fragments, using diff endonucleauses to cut DNA fragment |
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How is DNA sequencing automated? |
Dideoxy nucleotides fluorescently labelled analysed using a single capillary gel |
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Describe the process of genetic screening? |
Sequence mutated gene Produce DNA probe Clone probe using PCR Mix with sample (single-stranded DNA) idnetify those with probe |
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What is the importance of genetic screening? |
IMplications of having children Regular checks and earlier diagnosis take precautions/change lifestyle to reduce risk |
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How do you get a sample of DNA from blood? |
Use white blood cells |
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What principle does genetic fingerprinting work on? |
Hypervariable regions in DNA - non coding reptetitive base sequnces (core sequences), are more similar in more closely related people (number and length diff in diff people) |
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What is a minisatellite and a microsatellite? |
Mini is 10-100 base pair hypervariable region micro is 2-4 |
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Describe the process of genetic fingerprinting |
DNA is extracted isolated cut up using endonucleases amplified using PCR immersed in alkali to separate strands electrophoresis to separate strands southern blotting to transfer onto nylon membrane using capillary action hybridisation with DNA probe (binds to core seuqnece) legnth of core sequences compared in DNA profile |
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What are the uses of genetic fingerprinting? What are the issues? |
Identify how similar two individuals are forensic analysis (contamination/left at diff time to crime/relative) medical diagnosis (certain disease caused by length of allele/compare to those afflicted) Plant and animal breeding - prevent inbreeding/paternity test/see if desirable character Genetic variation |
