Manipulating genomes Flashcards

Question

Variable number tandem repeats

Answer 1

Short nucleotide sequence that is repeated throughout the genome the number of this varies at any given locus in the genome

Answer 2

closely related

Answer 3

Obtain the DNA = extracted from the root of a hair / spot of blood / semen / saliva Increase the quantity of DNA by using PCR Use restriction endonucleases to cut the amplified DNA molecules into fragments Separate the fragments using gel electrophoresis gel immersed in alkali - separate DNA double strands into single strands transferred onto a membrane by southern blotting Add radioactive or fluorescent probes in excess complementary + bind to specific VNTR regions - hybridisation X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing. These images contain patterns of bars (the DNA profile) which are then analysed

Answer 4

different restriction endonucleases - cut at different recognition / restriction sites make two cutes - once through each strand of DNA double helix

Answer 5

radioactive / fluorescent DNA probes added in excess to single stranded DNA fragments on the membrane bind to complementary strands of DNA

Answer 6

DNA (-ve) from gel electrophoresis is transfereed to a +vely charged membrane e.g. nylon Fragments are irreversibly bound to the blot, whilst maintaining their relative positions on the gel

Answer 7

Single stranded short piece of DNA with a known complementary sequence to the VNTR Synthesised chemically and is radio-labelled

Answer 8

X ray images of the membrane

Answer 9

membrane placed under UV light so fluorescent tags glow

Answer 10

Tissue typing = Donor + recipient tissues matches to reduce risk of rejection Detection of oncogenes = Can inform medication Detect mutations = E.g. – embryo selection Identify type of viral infection + monitor spread of infectious disease = PCR covid testing Identify suspects of crimes // forensic science = DNA profile of sample compared to samples from suspect / criminal database / victim = Identify bodies determine familial relationships for paternity cases species conservation to help scientists with captive breeding programmes to reduce chances of inbreeding

Answer 11

polymerase chain reaction

Answer 12

in vitro method of DNA amplification used to produce large quantities of specific fragments of DNA/RNA from very small samples even just 1 molecule of DNA / RNA

Answer 13

small sample of target DNA 2 primers Taq polymerase Free nucleotides Buffer Thermal cycler

Answer 14

Complementary to the 3’ end

Answer 15

One for each single strand of the now broken double helix

Answer 16

DNA polymerase from thermophilic bacteria in hot springs

Answer 17

does not denature at high temp

Answer 18

optimum pH for reactions beaker

Answer 19

automated provided correct temp for correct time

Answer 20

denaturation annealing elongation / extension

Answer 21

DNA fragments / primers / DNA polymerase / nucleotides added to thermocycler heated to 95°C breaks the hydrogen bonds forms 2 separate strands with exposed nucleotides denaturation – separating strands

Answer 22

temperature is decreased to between 50 - 60°C primers (forward and reverse ones) can anneal to the ends of the single strands of DNA

Answer 23

temperature is increased to 72°C for at least a minute optimum temperature for Taq polymerase to build the complementary strands of DNA produce the new identical double-stranded DNA molecules

Answer 24

Number of original DNA strands X 2^number of PCR cycles = total number of strands

Answer 25

very rapid – millions of copies made in hours does not require living cells – only base sequence PCR – needed for the sanger method + DNA sequencing

Answer 26

molecules are separated according to their size/mass and their net charge

Answer 27

electrical charge mass / size type of gel

Answer 28

negatively charged DNA - negatively charged due to the phosphate groups when placed in an electric field the molecules move towards the anode

Answer 29

Different sized molecules move through the gel at different rates tiny pores in the gel result in smaller molecules moving quickly + larger molecules slowly

Answer 30

Different gels have different sized pores affect the speed at which the molecules can move through them

Answer 31

Create an agarose gel plate in a tank Wells are cut into the gel at one end Submerge the gel in buffer solution Load the fragments into the wells using a micropipette Apply an electrical current to the tank. The smaller mass / shorter pieces of DNA fragments will move faster + further from the wells smallest fragments reach end - turn off electric current gel placed in alkaline buffer solution The fragments are not visible must be transferred onto absorbent paper or membrane via southern blotting fixed in place by UV / heat Probes are then added X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is generally compared to a control fragment of DNA

Answer 32

denature DNA fragments two strands separate

Answer 33

allow VNTRs / sequences to be identified A radioactive label which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands A fluorescent stain / dye which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands

Answer 34

Denaturing (to break the disulfide bonds) manipulating the proteins into rod shapes (negatively charged) to allow separation by size

Answer 35

nylon membrane

Answer 36

DNA sequence of COMPLEMENTARY STRAND

Answer 37

isolate the gene multiply DNA fragment - PCR formation of recombinant DNA transferring the vector

Answer 38

cut the DNA or isolate mRNA

Answer 39

restriction endonucleases cut 2 DNA fragments unevenly sticky ends – regions with unpaired / exposed bases make it easier to insert the desired gene into DNA of diff organism

Answer 40

blunt cuts vs sticky ends

Answer 41

isolating mRNA for gene – reverse endonucleases using reverse transcriptase produce single strand of complementary DNA - cDNA

Answer 42

easier to identify desired gene – particular cell will make very specific types of mRNA + mRNA + cDNA – no introns e.g. – beta cells in pancreas make insulin – a lot of insulin mRNA

Answer 43

multiply DNA fragment - PCR

Answer 44

form recombinant DNA

Answer 45

Same restriction endonuclease cuts plasmid Complementary sticky ends to DNA fragment Line up sticky ends DNA ligase forms phosphodiester bonds between sugar-phosphate groups

Answer 46

Usually bacterial DNA / plasmids

Answer 47

Replicate independently plasmid gets int host cell + combines with host DNA – form recombinant DNA Have marker gene e.g antibiotic resistance – can tell if bacteria have taken up gene by growing in media containing antibiotic

Answer 48

Plasmid – has second marker gene – show that plasmid contains recombinant DNA Restriction enzyme inserts desired gene in this marker gene If inserted right – marker gene wont function

Answer 49

transfer the vector

Answer 50

Plasmid with recombinant DNA – inserted into host cell (bacteria)

Answer 51

calcium solution or electroporation

Answer 52

Culture bacterial cells + plasmids in calcium rich solution Increase temp Causes bacterial membrane to become permeable + plasmids enter

Answer 53

Small electrical current applied to bacteria Membranes – become very porous + plasmids move in Also be used to get DNA fragments directly into eukaryotic cells DNA – pass through cell + nuclear membrane Control power or damage membrane

Answer 54

electrofusion

Answer 55

Tiny electric currents applied to membranes of 2 diff cells Fuses cells + nuclear membranes Forms hybrid / polyploid cell Contains DNA from both Produce GM plants Animal cells – do not fuse easily – membrane have diff properties Used in monoclonal antibodies

Answer 56

Agrobacterium tumefaciens – causes tumours in healthy plants Desired gene placed in the Ti plasmid + marker gene (antibiotic resistance / fluorescence) Carried into plant cell DNA Transgenic plant forms callus

Answer 57

mass of GM plant cells

Answer 58

expose host to an antibiotic that kills cells that lack the new genes wait for surviving cells to form a callus

Answer 59

Electrofusion Remove plant cell wall by cellulases Electrofusion to form new polyploid cell Plant hormones to stimulate growth of new cell wall Callus formation Transgenic plant formarion

Answer 60

organism contains nucleotide sequences from a different species

Answer 61

universal, meaning that almost every organism uses the same four nitrogenous bases same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species) mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism

Answer 62

Plasmids - transfer DNA into bacteria or yeast Viruses - transfer DNA into human cells or bacteria Liposomes - fuse with cell membranes to transfer DNA into cells

Answer 63

class of enzymes found in bacteria used as a defence mechanism bagainst bacteriophages (viruses that infect bacteria) e enzymes restrict a viral infection by cutting the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule.

Answer 64

Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome

Answer 65

DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion

Answer 66

Microinjection - injecting the plasmid Heat shock w/ calcium salts Electroporation Electrofusion

Answer 67

Reducing the temp to freezing and rapidly increasing to 40 degrees - increases permeability Ca^2+ surrounds DNA (-ve), reduces repulsion, increases permeability

Answer 68

Reproduce asexually - no genetic variation Taking up plasmids from surroundings increases genetic variation, allows selection and evolution

Answer 69

these cells will carry out the post-translational modification - due to the presence of Golgi Apparatus

Answer 70

More cost-effective to produce large volumes Faster to produce Reliable supply available engineered to be identical to human proteins / have modifications that are beneficial moral or ethical or religious concerns against using cow or pork produced proteins Less allergic reactions

Answer 71

Bacteria plasmids modified to include human insulin gene Inserted into E coli via transformation Identified + isolated transgenic bacteria Express human protein insulin Extracted + purified

Answer 72

Better than selective breeding Organisms with the desired characteristics are produced more quickly All organisms will contain the desired characteristic The desired characteristic may come from a different species/kingdom

Answer 73

Resistant to herbicides – increases productivity / yield Resistant to pests – increases productivity / yield Enriched in vitamins – increases the nutritional value Golden rice reduce the impact of farming on the environment due to there being less need to spray pesticides

Answer 74

soya beans – susceptible to insect pests genetically modified the already herbicide-resistant variety of soybean) by inserting a gene for the Bt toxin gene is taken from the bacterium Bacillus thuringiensis produce their own insecticide insect ingests parts - alkaline conditions in their guts activate the toxin the toxin is harmless to vertebrates as their stomach is highly acidic killing the insect BUT insect populations developed resistance

Answer 75

produce pharmaceutical drugs – pharming biopharma sheep and goats have been genetically modified to produce a number of useful human proteins in their milk the human blood protein known as AAT in sheep milk the human protein antithrombin (stops blood clotting) in goat milk

Answer 76

modified to shed light on their metabolism, drug resistance as well as how it causes damage to its host aid research act as vectors – infect cells – modified so can not replicate when inside host cell

Answer 77

Biotech companies charge farmers more money for GM seeds vs non-GM seeds to try and make back the money they have invested in their product Seeds can not be kept from GM crops to regrow the crop the following year because GM crops do not "breed true" Buying seeds year upon year can be a major struggle for farmers in developing countries – only buy from patent holder lack of long-term research on the effects on human health Organic farmers have complained that the pollen from GM crops may contaminate nearby non-GM crops that have been certified as organic Environmentalists are concerned about the reduction in biodiversity for future generations more vulnerable to extinction Herbicide-resistance genes could transfer to weed plants resulting in "superweeds" antibiotic-resistance genes that are commonly used as marker genes in genetic engineering could transfer to pathogenic organisms that would then be untreatable with antibiotics - "superbug" Patenting – people in less developed countries prevented from using GM crops by patents // unable to afford

Answer 78

involves using various mechanisms to alter a person's genetic material to treat, or cure, diseases

Answer 79

* replacing mutant allele with healthy allele in affected somatic cell

Answer 80

Ex vivo – the new gene is inserted via a virus vector into the cell outside the body. Blood or bone marrow cells are extracted and exposed to the virus which inserts the gene into these cells. These cells are then grown in the laboratory and returned to the person by an injection into a vein In vivo – the new gene is inserted via a vector into cells inside the body

Answer 81

insert healthy allele into germ cell – egg / embryo immediately after fertilisation

Manipulating genomes Flashcards

(110 cards)