6.3 MANIPULATING GENOMES Flashcards

Question

What are the uses of DNA profiling?

Answer 1

Uses of DNA profiling: - maternity and paternity disputes - forensic science = convictions of crimes, proving innocence, identify victims body parts, identify descendants of soldiers killed in WWII, identify Nazi war criminals in South Africa, used in immigration cases to prove or disprove family relationships

Answer 2

The agarose gel is placed into a buffering solution to maintain constant pH.

Answer 3

DNA ladders show known base pair fragment distances so unknown fragments can be compared to the DNA ladder and identified

Answer 4

DNA molecules move towards the anode in an electrophoresis tank as DNA molecules are negatively charged (due to the negative charge on the phosphate group) so they are attracted to the oppositely charged cathode

Answer 5

Within agarose gel, the size of the DNA fragment will determine how far it travels. Due to the 'mesh like' property of the agar gel, small fragments travel further.

Answer 6

When the gel has finished running it is placed into an alkaline buffer to denature the alkaline DNA and expose its bases.

Answer 7

Southern blotting is when strands are transferred to nitrocellulose paper/nylon membrane. The membrane is covered with several sheets of dry absorbent paper which draws alkaline solution (containing DNA) through the membrane.

Answer 8

To identify certain genes that may cause genetic disorders, we can use DNA probes and DNA hybridisation.

Answer 9

Uses of DNA probes: DNA probes are small fragments of nucleic acids (either cloned or artificially synthesised) that is labelled with an enzyme, a radioactive tag or a fluorescent tag fluorescent dye tag - shows up as fluorescent bands when gel is exposed to a UV light source radioactive tag - shows up as a dark band when the gel is exposed to photographic film - the probe will bind to a complimentary DNA sequence by base pairing, identifying the presence and location of the target DNA sequence for further analysis - a DNA probe is 50-80 nucleotides long made with complimentary bases to the gene of interest - DNA strands are separated - separated strands are mixed with DNA probe - DNA probe binds to complimentary bases - location can be identified by radioactivity/fluorescence of DNA sample - need to know the base sequence of the gene first or presence can be identified

Answer 10

Genetic screening: - many genetic disorders are the result of a mutation - if the mutation occurs in a dominant allele, all individuals who possess this allele will have the disorder - if the mutation occurs in a recessive allele, heterozygous individuals will be carriers of the disease, they could pass the disease to their offspring if their partner is also heterozygous or homozygous recessive - genetic councillors advise parents of the likelihood of their children having the disease . uses family history of a genetic disease

Answer 11

Sanger sequencing: - four different terminators used (A, C, T and G). each terminator is a modified nucleotide that cannot polymerise to a subsequent nucleotide - four reactions are run, in each reaction you have the DNA being sequenced, a mixture of 'normal nucleotides', one type of terminator nucleotide, a primer, DNA polymerase - each tube contains millions of copies of the DNA template, nucleotides, and a good supply of the specific terminator nucleotide - due to this, you get a variety of 'partially completed' DNA strands, because they have been 'terminated' at different points

Answer 12

Sanger sequencing now: - terminator nucleotides are labelled with fluorescent dyes (instead of radioactive isotopes) - the fragments are run through a single long thin tube containing a gel matrix in a process called capillary gel electrophoresis - short fragments move quickly and long fragments move more slowly - at the end of tube, its illuminated by a laser, allowing the attached dye to be detected

Answer 13

Uses and limitations of Sanger sequencing: - typically used to separate pieces of DNA such as plasmids or DNA copied in PCR - however, this method is expensive and inefficient for larger scale projects - new large scale sequencing techniques are faster and less expensive = next generation sequencing - the human genome can now be sequenced in a day

Answer 14

Next generation sequencing advantages: - highly parallel = many sequencing reactions take place at the same time - fast = because reactions are done in parallel, results are already much faster - low cost = sequencing a genome is cheaper than with Sanger sequencing

Answer 15

Comparing species: - we share 94-99% of our DNA with chimpanzees - genes that work well are conserved by evolution e.g humans and pigs have very similar genes for insulin

Answer 16

DNA barcoding identifies sequences of a genome common to all species but which also have some variation- these are called conserved sequences - iBOL (international barcode of life) identify species using relatively short sections of DNA = in most animals - a 648 base pair region called co1 in mDNA which codes for cytochrome c = quick and cheap to sequence as relatively small = not effective for identifying plants as they don't vary enough in that gene - for land plants, 2 regions in chloroplast DNA have been approved - cannot be used yet for fungi or bacteria (no suitable gene formed)

Answer 17

Evolutionary relationships: - all organisms share common ancestors - comparing genomes tells us how closely related they are = because we know the basic mutation rate scientists can calculate how long ago two organisms diverged from a common ancestor - evolutionary trees are more accurate as a result

Answer 18

Proteomics and genomics: - proteomics investigates how proteins affect and are affected by cell processes or the external environment - within an individual organism, the genome is constant, but the proteome varies and is dynamic - every cell in an individual organism has the same set of genes, but the set of proteins produced in different tissues differ from one another and are dependent on gene expression

Answer 19

Proteomics is the branch of molecular biology that studies the set of proteins expressed by the genome of an organism.

Answer 20

A proteome is the complete set of proteins encoded by a particular genome.

Answer 21

Genomics is the study of the complete genome of an organism.

Answer 22

Spliceosomes: - mRNA transcribed from DNA includes both the exons and introns - before lining up on ribosomes pre mRNA is modified (introns removed and in some cases some exons are removed too) - the exons to be translated are joined together by enzyme complexes known as spliceosomes to give the mature functional mRNA - spliceosomes may join the same exons in a variety of ways = as a result, a single gene may produce several versions of functional mRNA, which would code for different arrangements of amino acids, giving different protons and resulting in different phenotypes

Answer 23

Protein modification: - some proteins are modified by other proteins after they are synthesis - a protein that is coded for a gene may remain intact or it may be shortened or lengthened to give a variety of other proteins

Answer 24

The development of software and computing tools needed to analyse and organise raw biological data

Answer 25

Computational biology is the study of biology using computational techniques to analyse large amounts of data. Uses bioinformatic data to build theoretical models of biological systems, which can be used to predict what may happen in different circumstances.

Answer 26

A genotype is the genetic makeup of an organism

Answer 27

A phenotype is observable characteristics of an organism

Answer 28

Epidemiology is a branch of medicine which deals with the incidence, distribution and possible control of diseases and other factors relating to health

Answer 29

Synthetic biology is the design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems.

Answer 30

Importance: - sequencing billions of base pairs in DNA - working out 3D structures of proteins - understanding molecular pathways such as gene regulation

Answer 31

Use information from gene sequencing: identifying genes linked to specific diseases in populations and in determining the evolutionary relationship between organisms

Answer 32

Analysing and comparing genomes can reveal patterns in DNA we inherit and diseases we are vulnerable to

Answer 33

Genomics is changing the face of epidemiology- we can now study incidence, distribution, and control of disease. personalised medicine- appropriate drugs for genetic cause e.g herceptin for breast cancer

Answer 34

Scientists have been able to recognise that our genes work together with the environment to affect our characteristics, physiology and likelihood of developing diseases

Answer 35

Sequencing genomes has allowed: - doctors to find the source of an infection - doctors to identify antibiotic-resistant strains of bacteria - genome analysis makes it easier to track the spread of transmission and to plan treatment - scientists to track the progress of an outbreak of potentially dangerous disease and monitor potential epidemics. this has implications for successful treatment of potentially fatal diseases - scientists to identify regions of a pathogens genome that can be used as targets for new drugs and vaccine development.

Answer 36

Recombinant DNA is DNA joined from 2 or more sources by genetic engineering.

Answer 37

A transgenic organism is an organism that has been genetically altered by the introduction of foreign DNA from another species by artificial means.

Answer 38

Genetic engineering is the extraction (or manufacture) of a gene from one organism and insertion into another to cause gene expression

Answer 39

Restriction enzymes bind to and break DNA at specific sites.

Answer 40

Blunt ends are a cut straight across the double helix.

Answer 41

Genetic engineering process: 1. identification of a desired gene in one organism 2. removal of gene from DNA (restriction endonuclease leaves 'sticky ends' 3. inserting the new into the plasmid (DNA ligase enzyme) 4. putting the plasmid into the bacterial cell is called transformation. only a few of the bacterial cells take up the plasmids 5. transgenic organism can be cloned to produce identical copies in a fermenter 6. desired products are made e.g insulin

Answer 42

Restriction endonuclease are enzymes that cut DNA at certain recognition points. A wide variety of them exist which create different sticky ends

Answer 43

Sticky ends are single stranded sections of DNA with unpaired bases that can base pair with complimentary sticky ends

Answer 44

DNA ligase is an enzyme that forms phosphodiester bonds between the sugar and phosphate groups of the two strands of DNA, joining them together

Answer 45

Reverse transcriptase is an enzyme that synthesises DNA from the RNA (reverse of transcription), retroviruses have RNA. allows us to generate DNA (a gene) from mRNA- pre HGP

Answer 46

Reverse transcriptase: 1. find a cell that produces the protein you require e.g beta cells of islets of langerhans in the pancreas to produce insulin 2. extract the mRNA from the cells 3. use reverse transcriptase to make DNA from the RNA, now called complementary DNA, cDNA (made up of complimentary bases to the mRNA) 4. the 2nd strand is made using DNA polymerase 5. gene for insulin is produced

Answer 47

Antibiotic-resistant markers: - bacterial plasmid has two genes for antibiotic resistance - the insulin (or desired gene) is inserted into the plasmid midway through the gene for tetra resistance - some plasmids take up the gene, some do not - only bacteria that have taken up the plasmids survive on the AMP plate - bacteria only grow on the tetra plate if they have not got the insulin gene

Answer 48

Fluorescent markers: - plasmids are used that contain the gene for green fluorescent protein (GFP) - the desired gene (insulin) is added into the gene for GFP - if the bacterial colony is fluorescent it does not contain the desired gene

Answer 49

Enzyme markers: - desired gene is inserted into the gene for an enzyme (e.g lactase) - lactase enzyme turns a colourless substrate blue. the bacteria that remain clear on the substrate have the desired gene

Answer 50

How to get DNA into a cell: Electroporation- an electrical current is applied to the bacterium causing the membrane to become porous to allow the plasmid to pass through - also can be used to get small DNA fragments into some eukaryotic cells Electrofusion- two cells are stimulated by a small electric current in order to fuse them together to make one hybrid or polyploid cell - more successful in plants than animals as it is more normal for plant cells to hybridise and become polyploid, which is not ok for animals

Answer 51

GM soya (genetically modified): - beans produce Bt protein that is toxic to pest insects that attack it- farmers do not need to use pesticides - resistant to weed killer so that farmers can spray to get rid of weeds without affecting the crop

Answer 52

GM crops: pest resistance + reduce pesticide spraying, protecting env, helps poor farmers, increases yield - toxins in plant may affect non-pest insects and insect eating predators, insect pests may become resistant disease resistance + crops become resistant to common plant diseases, reduces crop losses/increases yield - transferred genes may spread to wild populations e.g super weeds herbicide resistance + reduce competing weeds, increase yield - biodiversity could be reduced if herbicides overused to destroy weeds, fear of super weeds extended shelf life + reduces food waste - reduce commercial value and demand for the crop growing conditions + crops grow in a wider range of conditions e.g flood and drought resistance nutritional value + can be increased- addition of vitamins etc - people may be allergic to different proteins made by GM crops medical uses + plants could be used to produce medicines or vaccines

Answer 53

GM plants ethics: - LICs may be prevented from using them by patents* and issues of technology transfer - those in need most may not be able to afford it *legal patent- for new technology/invention prevents people using something without payment. farmers cannot save seeds for the following year, they must buy them every year

Answer 54

GM pathogens for research: - GM microorganisms (usually bacteria/yeast) can be used to store a living record of DNA of another organism. (source of DNA fragments) - pathogens are sometimes GM for medical and epidemiological research. (under very regulations as could be used in warfare)

Answer 55

GM animals: - GM animals, especially vertebrates, are a tricky target for GM; some success inserting DNA within modified viruses or microinjections (particles of gold covered in DNA) e.g - swine flu resistance from wild African pigs into European pigs - fast-growing gene from Chinook salmon inserted into Atlantic salmon (in US) so they grow to adult size in half time of non-GM salmon

Answer 56

Pharming: - creating human proteins - human gene can be inserted into fertilised egg (cow/pig/sheep) - promotor sequence could mean the gene is only expressed in the mammary glands- milk will then contain the required human protein

Answer 57

Pharming for proteins: 1. desired gene cut out of cell in host species 2. a body cell is removed from party A and the nucleus is removed (enucleation) 3. the nucleus and desired gene are fused together 4. an egg cell is taken from party B and enucleated 5. the egg cell and nucleus containing the desired gene are fused 6. the egg is stimulated to divide to form an embryo 7. the embryo is put into the uterus of party C (surrogate) 8. surrogate gives birth to transgenic organism (party D)- DNA contains the desired gene 9. desired gene can be extracted from milk of party D e.g goats and spider silk

Answer 58

GM animals and microorganisms and ethical issues: GM E.coli producing human insulin - diabetics receive human insulin, reducing human pain and suffering GM pigs that express higher levels of growth hormone - pigs grow too fast, causing heart discomfort when exercising GM goats that produce human lysozyme - protects humans who drink the goat milk from diarrhoea planning to clone northern white rhino - prevent extinction of northern white rhino cloned cattle - increased susceptibility to disease in cattle children born with SCID treated with gene therapy - children cured of SCID, removing pain and suffering GM 'oncomouse' patented - patented animals as human property

Answer 59

GM crops and ethical issues: GM rice contains beta carotene genes - people who eat GM rice less likely to suffer from vitamin A deficiency GM tomatoes that take longer to soften after ripening - concerns over safety of eating GM food GM maize that is resistant to drought - greater yield of maize in countries with drought, feeding more people and averting famine GM soya contains gene that makes it resistant to insects - if GM soya crossbreed with wild plants, resistance gene could be spread to weeds GM tobacco plants that produce therapeutic antibodies - therapeutic antibodies used to treat rabies and save human lives patented GM seeds - farmers cannot use seed from crops and must buy more seed from the manufacturer

Answer 60

Gene therapy is the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders.

Answer 61

Germ line cell gene therapy: - the corrected gene is inserted into a fertilised egg produced via IVF - if successful, all cells of the embryo will contain the corrected gene when the cell divides by mitosis - germ cell therapy is permanent and also ensures offspring inherit corrected gene - currently illegal

Answer 62

Somatic cell gene therapy: - copies of the corrected gene are inserted directly into the somatic (body) cells of the sufferers - this type of gene therapy does not prevent the disease occurring in the next generation because it does not affect the sperm and egg cells - has to be repeated many times as the effects do not last long

Answer 63

Liposomes as vectors: 1. functioning genes isolated from human cells and inserted into plasmids 2. plasmids extracted and wrapped in lipid molecules (forming a liposome) 3. liposomes sprayed into patients airways via a nasal aerosol 4. liposomes pass easily through the plasma membrane of cells and nucleus

Answer 64

Viruses and vectors: 1. grow harmless adenoviruses in epithelial cells in a lab 2. add recombinant plasmids that contain functional gene 3. viruses isolated from epithelial cells and purified 4. viruses sprayed into the nostrils of patients via aerosol 5. virus injects DNA into epithelial cells of patients' lungs

Answer 65

Genetically engineering plants: 1. the plasmid is removed from the bacterium and the T-DNA is cut by a restriction enzyme 2. foreign DNA is cut by the same enzyme 3. the foreign DNA is inserted into the DNA of the plasmid 4. the plasmid is reinserted into a bacterium 5. the bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of the plant cell 6. the plant cells are grown in culture 7. a plant is generated from a cell clone. all of its cells carry the foreign gene and may express it as a new trait

Answer 66

PCR is used to produce many copies of a small piece of DNA in a range of sizes.

Answer 67

Electrophoresis is used to order DNA fragments by size using an electric current to sequence the bases.

Answer 68

Digestion of DNA by restriction enzymes is used to cut the genome into smaller fragments and cut vectors for the gene library.

Answer 69

A genome has to be fragmented before sequencing as they are very large/too large to be sequenced whole and smaller fragments allow for a better accuracy.

Answer 70

Vitamin A rice: . benefits - reduced vitamin A deficiencies in Asia where rice is already a staple diet - reduced blindness . concerns - reduces the genetic diversity of rice - hybridisation with wild rice

6.3 MANIPULATING GENOMES Flashcards

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