MANIPULATING GENOMES 6.1.3 OCR A LEVEL BIOLOGY

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  • Created by: Davina1st
  • Created on: 21-11-21 15:11
What is a genome?
All of the genetic material an organism contains. E.g. in eukaryotes the dna and mitochondria combined.
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What are exons/introns?
Exons- parts of DNA that code for proteins only 2%. Introns-large non-coding parts of DNA, in between genes, transcribed along with genes and removed after transcription.
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What are variable number tandem repeats? (VNTR's/minisatellite)
A location in a genome where a short nucleotide is organised as a tandem repeat. These can be found on many chromosomes and often show variations between individuals. 10-100 base pairs long, repeating 50-several hundred times.
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What are short tandem repeats? (microsatellite)
Smaller repeated sequences within non-coding dna 2-9 base pairs long repeated 5-15 times.
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Satellites appear in the same position on chromosomes, and vary between individualstrue or false?
True
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What is DNA profiling used for?
To produce images of the patterns in DNA, used to assist in the identification of individuals and familial relationships.
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What are the 5 steps of DNA profiling?
Extract the DNA, digest the sample with enzymes, separation of DNA fragments (gel electrophoresis), hybridisation, making it visible
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Describe extracting the DNA (DNA profiling)
Extracted from mouth, saliva, blood, hair or bone. DNA purified so histones are removed by protease enzymes (so DNA cane be sequenced). Only small amounts needed, as it can be replicated to give larger samples.
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Describe how the sample is digested using enzymes in DNA profiling
DNA cut into smaller fragments using restriction enzymes, which cut at specific base sequences. Particular base sequence of DNA=specific complimentary active site of enzyme. Relevant bond broke (H bonds and phosphodiester).
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What do restriction enzymes do?
Cut DNA at specific base sequences leading to blunt or staggered ends.
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Describe how DNA fragments are separated in DNA profiling
Electrophoresis,DNA fragment placed in well of gel block and stained. Placed in alkaline buffer solution =regulate ph and carry electric charge. Electric current passed through gel. fragments move to positive end.
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Why do DNA fragments move to he positive end during electrophoresis? What does it mean when some fragments travel further than others?
Phosphate groups in DNA have a negative charge, the smaller the fragment the further it moves due to less resistance.
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After the electric current is passed through the gel block during electrophoresis, what happens?
DNA made single stranded ready for analysis. Nylon membrane placed on top of gel, with absorbent paper on top,, this draws top starnds of FNA onto the nylon = southern blotting. DNA is transferred onto the sheet in exact same place as on the gel.
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How are DNA strands fixed onto the nylon sheet after electrophoresis/southern blotting?
By UV light or heat at 85 degrees Celsius.
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Describe the process of hybridisation in DNA profiling
DNA probes added to look for micr/mini satellites. /when the probe attaches to the sequence you are looking for, it is called hybridisation. Any excess probes that are not attached are washed away, so not to be detected.
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What is a DNA probe?
Single stranded DNA used to detect the presence of a complimentary strand of DNA by hybridisation.
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How is the DNA made visible during DNA profiling?
If radioative probes are used, it can be seen when they attach, if radioactive probes were used, placing the probes on the nylon membrane causes the film to go darker where the probe is. If fluorescent, nylon membrane under UV light, probes will glow
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Why are many different short tandem repeats looked for, instead of just 1?
There is a chance that that 2 people could have the same pattern for one particular STR in their DNA but the chances of having the same DNA pattern for 12 different STR's is very unlikely.
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How is a small DNA sample amplified to make many copies?
By PCR (polymerase chain reaction)
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What is needed for PCR?
Excess of free nucleotides needed, original sample of double stranded DNA, primers (short pieces of single stranded DNA which start the copying process), they are 15-35 nucleotides long, DNA polymerase enzyme.
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What are the three stages of PCR?
Denaturing stage, annealing stage and extending stage.
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What happens in the denaturing stage of pcr?
Temp inside the machine is 90-95°C for 30 seconds. This breaks the H bonds between the 2 strands of DNA, separating them.
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What happens during the annealing stage of pcr?
Temp decreased to 55-60°C, primers anneal (join) to both ends of the single stranded DNA, complimentary to the ends of the DNA which is needed for replication to start.
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What happens during the extension phase of pcr?
Temp increased to 70-75°C for 1 minute, which is optimum temperature for chosen DNA polymerase enzyme to work, this adds bases to the primer, extending the complimentary strand = double stranded DNA genetically identical to sample.
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Why might you not get as many copies of DNA as expected using pcr?
Not enough primers present, primers may not attach to all the DNA strands, may be insufficient nucleotides available, when 2 DNA strands separated, they may re-join to each other instead of the primers.
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What is the difference between a DNA primer and a DNA probe?
Probes are larger (100-1000 nucleotides long) whereas a primer is 15-25 nucleotides long and is used to start the replication on DNA strands during PCR.
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Give 4 uses of DNA profiling
Forensic science/criminal investigations, proving paternity of a child, immigration cases to prove/disprove familial relations and identifying individuals at risk to a specific disease.
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What is DNA sequencing?
The process of determining the nucleic acid sequence - the order of nucleotides in DNA, includes any method or technology that is used to determine the order of the 4 bases.
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What is the human genome project?
HGP was an international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA, and identifying and mapping all the genes of the human genome from both a physical + functional standpoint
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what is the difference between DNA profiling and DNA sequencing?
DNA profiling only looks for specific sequences within DNA, whereas DNA sequencing looks at the specific order of nucleotides/base pairs.
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What are terminator bases?
A-green, G-yellow, T-red, C-blue, modified versions of base pairs with fluorescent markers, doubly de-oxidised
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Describe the process of DNA sequencing
Multiple copies of DNA made using pcr, DNA broken into shorter pieces to make analysis easier, made single stranded by heat and PCR conducted, each DNA is copied until point where terminator joins=different lengths of DNA
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What is the next stage after different lengths of DNA is produced by DNA sequencing?
The lengths of DNA are separated by capillary tube electrophoresis, shorter lengths travel further, computer records the colours as they pass out of the end of the capillary tube. First colour represents the base at the end of the shortest fragment.
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What is next generation sequencing/whole genome sequencing?
Also known as high throughput sequencing, generates and analyses millions of sequences per run, allowing researchers to sequence, resequencing and compare data at a rate previously not possible.
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What is an advantage of next gen sequencing?
Much faster and more affective, computer technology allows the whole genome to be sequenced = lowered costs.
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What has gene sequencing allowed for?
Genome comparisons between individuals in a species and between members of different species, allowed for sequence of amino acids in proteins to be predicted, development of synthetic biology
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What is the basis of genetic engineering?
Isolating a gene for a desirable characteristic, and placing it into another organism using a suitable vector, an organism that carries a gene from another organism is often called 'transgenic' or GMO
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What is a promotor? genetic engineering
A region of DNA that initiates transcription of a particular gene. Promotors are located near the transcription start sites of genes.
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When a gene is transferred to another organism, the promotor sequence is not needed, true or false?
False, the promotor is needed so that the gene can be transcribed.
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Give some examples of reasons for genetic engineering
Making crops resistant to pests and diseases, making livestock resistant to pests and diseases, improving the yields from plants and livestock, improving nutritional qualities of crops. Modifying animals to make human products for medicine.
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What are the 4 stages of genetic engineering?
Required gene is obtained, a copy of the gene is placed inside a vector, the vector carries the gene into a recipient cell, the recipient expresses the novel gene.
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What are the three ways in which the required gene can be obtained for genetic engineering?
Using mRNA, using a DNA probe or using an automated machine.
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How can you obtain the required gene for genetic engineering using mRNA?
Obtain the mRNA for that gene in cell where that gene is expressed, add free DNA nucleotides+reverse transcriptase which turns mRNA into single strand of DNA (cDNA). Single strand of DNA made double stranded-DNA nucleotides, primers, DNA polymerase.
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How can you obtain the required gene for genetic engineering using a DNA probe?
Make the DNA single stranded by heating, then add probe which is complimentary to the gene you are looking for, once the gene is found, it can be cut out using restriction enzymes. (probe - fluorescent, radioactive stranded piece of DNA)
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How can you obtain the required gene for genetic engineering using an automated machine?
If you know the base sequence of the gene, the gene can be synthesised artificially in the lab, by adding DNA nucleotides and DNA polymerase andprovide a suitable temp.
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What is a vector in genetic engineering?
'Gene carriers' e.g. plasmids from bacterial cells, viruses as they naturally insert their DNa into the host, Ti plasmids which are from soil bacteria and genetically modified plant cells.
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How is a gene inserted into a vector?
Restriction enzymes are used to cut open the vector DNA and to cut the required gene out of the DNA it is attached to.
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What are restriction enzymes? Genetic engineering
Recognise specific base sequences and within DNA and will cut DNA at those points, recognition site: 4-6 bp long, usually palindromic(reads the same both ways), some of them leave blunt ends while others leave staggered ends.
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Are staggered ends or blunt ends more useful?
Staggered, as if the two pieces of DNA are cut with the same restriction enzyme, the sticky ends will have complimentary unpaired bases which can be joined A=T C≡G
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What does DNA ligase do? What is recombinant DNA?
DNA ligase, joins the complimentary sticky ends (joins their sugar phosphate backbones). When the two pieces join, it is called recombinant DNA.
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Why are marker genes used in genetic engineering?
Allows the gene that you want to be tracked, so you can check if it has been taken up by host cell. You need to know which cells are successfully modified before growing on a large scale. Often resistance to antibiotic/fluorescent.
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How do you get the vector into the recipient cell in genetic engineering? Which techniques are used?
Recombinant plasmids added to solution of bacterial host cells, DNA doesn't easily cross membranes. Heat shock treatment: alternating hot and cold with CaCl ions=porous walls. Electroporation=high voltage impulse to disrupt csm. Electrofusion
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How can you identify which cells have been successful in taking up the new gene in genetic engineering?
If bacteria has antibiotic resistance (from marker gene) and no fluorescence that means they have taken up the plasmid and the gene. These can then be replicated to make more of the gene.
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Why is bacteria usually genetically engineered instead of prokaryotes?
Simple cells-easy to remove plasmid, modify and put back, fast growing-reproduce asexually by cloning themselves=millions of modified cells, no ethical issues, cheap to grow.
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How can plants be genetically modified?
Desired gene is placed into plasmid from agrobacterium with marker gene, plasmid inserted back into bacterium and added to dish containing plant cells, plasmid passes directly into plant cells=callus which can grow into a separate plant.
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How are polyploids formed? genetic engineering
2 different plant cells fused together by tiny electric currents = hybrid cell (cell and nuclear membranes fused together). The hybrid cell is polyploid as it has more than 2 sets of chromosomes.
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What are some advantages of genetic engineering compared to selective breeding?
Can alter single specific characteristics rather than crossing two whole genomes, much quicker results than selective breeding (it is seen in one gen), can transfer genes between different species of animals and plants which is normally impossible.
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What are some disadvantages of genetic engineering compared to selective breeding?
Unknown side effects of transferring genes between species, often seen as unethical by the public, expensive procedures with low success rates.
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What is gene therapy?
The introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. Switching off/silencing a harmful gene/allele.
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How does gene therapy change depending on if the genetic disorder is caused by two recessive alleles, or by a dominant allele?
2 recessive- working copy of dominant allele introduced into the cell which can mask the two fault versions. Dominant - allele not removed, just silenced e.g. sticking another section of DNA in middle of faulty allele so can no longer be transcribed.
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What does somatic cell gene therapy include?
Replacing mutant allele with a healthy allele in somatic body cells. Viral vectors are often used. Or liposomes like in cystic fibrosis.
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How are viruses used as vectors in gene therapy?
Cells from patient harvested, virus is altered so it cant reproduce, gene inserted into virus, altered virus mixed with patients cells, cells become transgenic, altered cells injected into patients body which produce the desired protein.
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What are some potential problems of using viruses as gene vectors? in gene therapy
May stimulate immune/inflammatory response, patient may become immune to the virus, so it can be used to deliver that allele, virus might insert the allele into human DNA in a region that disrupts the expression of other genes.
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What does germ line cell gene therapy involve?
Altering the alleles of gametes or an egg immediately after fertilisation as part of IVF, which means all the cells offspring would have the genetic modification (passed onto offspring) Possible but currently illegal.
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Why is germ line gene therapy currently illegal?
Potential impact to resulting offspring is unknown, no consent given by unborn offspring, it is irreversible, could enable people to choose desirable characteristics for their unborn children.
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Why is somatic gene therapy only a temporary cure for disease?
Newly inserted correct allele will be passed on every time the cell divides by mitosis but somatic cells have a limited lifespan, have to be replaced by stem cells (which have original faulty allele) so after a while this is expressed again.
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What are the positives of gene therapy?
Prolonged life expectancy, better quality of life, temporary cure rather than ongoing treatment.
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What are the negatives of gene therapy?
Very expensive and resources could be better spent elsewhere, effects are only short lived, difficult to deliver allele to specific location where it is needed, could trigger immune response if viral vector is used.
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How have microbes been genetically modified?
Some tumour cells have receptors for poliovirus, so poliovirus will recognise and attack them. By genetically engineering poliovirus to inactivate genes that cause poliomyelitis, scientists can attack cancer cells without causing disease=treatment.
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What are some positives and negatives of genetically modifying pathogens?
Positive-previously untreatable diseases can be treated. Neg- scientists investigating pathogens could become infected/pathogen reverts back to normal form=mass outbreak of disease. In the wrong hands could be used as biowarfare.
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What are some positive and negative ethical issues regarding genetically modifying plants?
+ increased nutritional value, extended shelf life=reduced food waste, disease resistance - people may be allergic to GM plants, extended shelf life might reduce commercial value/demand, transferred genes may move to wild populations cause problems
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Give an example of genetic engineering in plants. (soybean plants with resistance to insect pests)
GM soybeans contain Bt bacteria toxic to pests. Taken up by plasmid containing the gene.
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Give an example of how animals have been genetically engineered. (animas to make pharmaceutical products)
DNA that codes for human antithrombin injected into goat, selective breeding of offspring with the desired gene leads to a herd of goats who produce antithrombin in their milk. Protein taken from milk and made into drug.
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What are some positive and negative ethical issues regarding pharming animals?
+ large quantities of drugs made more available to people - may have harmful side effects on animal, enforces the idea animals are merely 'assets' and can be treated however we choose.
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What are some ethical issues relating to patenting and technology transfer?
LEDC countries prevented from using GM due to patents in technology transfer, owner of patent gets money when product is used, farmers in poorer countries don't benefit.
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Other cards in this set

Card 2

Front

What are exons/introns?

Back

Exons- parts of DNA that code for proteins only 2%. Introns-large non-coding parts of DNA, in between genes, transcribed along with genes and removed after transcription.

Card 3

Front

What are variable number tandem repeats? (VNTR's/minisatellite)

Back

Preview of the front of card 3

Card 4

Front

What are short tandem repeats? (microsatellite)

Back

Preview of the front of card 4

Card 5

Front

Satellites appear in the same position on chromosomes, and vary between individualstrue or false?

Back

Preview of the front of card 5
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