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Biology Module 6 > 6.1.3 Manipulating Genomes > Flashcards

6.1.3 Manipulating Genomes Flashcards

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1
Q

what is PCR

A

polymerase chain reaction

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2
Q

what is the use of PCR

A
  • selecting a fragment of DNA with gene or bit of DNA you are interested in
  • and amplifying it into millions of copies
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3
Q

what is the reaction mixture used in PCR made of

A
  • DNA sample
  • free nucleotides
  • primers
  • DNA polymerase
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4
Q

what are primers

A

short pieces of DNA that are complementary to the bases at the start of the fragment you want

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5
Q

what is DNA polymerase

A

an enzyme that creates new DNA strands

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6
Q

what is the first step of PCR with temperature included

A
  • DNA mixture is heated to 95°C
  • breaking the hydrogen bonds between 2 strands of DNA
  • (DNA polymerase still doesn’t denature here, which is important, as it means many cycles of PCR can be carried out, and you don’t need to use a new temperature each time)
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7
Q

what is the second step of PCR with temperature

A
  • mixture is cooled to 50-60°C
  • means that primers can bind (anneal) to the strands
  • needed for replication to occur
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8
Q

what is the third step of PCR with temperature

A
  • the reaction mixture is raised to 72°C
  • optimum temperature for DNA polymerase to work
  • the DNA polymerase lines up free DNA nucleotides alongside each template strands
  • complementary base pairing means that new complementary strands form
  • enzyme used is Taq polymerase (used in hot springs, so can deal with high temperature)
  • 2 new copies of the fragment of DNA are formed
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9
Q

describe the mathematical replication during PCR

A
  • after one cycle, 2 new copies are made
  • the cycle starts again, and all 4 strands (2 new and 2 old) are used as templates
  • each PCR doubles the amount of DNA
    ( from 2 original to 4,8,16)
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10
Q

what is electrophoresis

A

procedure that uses electrical current to separate out DNA fragments, RNA fragments or proteins depending on their SIZE

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11
Q

what is the first step of gel electrophoresis, where the gel tray is added to the gel tank/box

A
  • often performed using agarose gel that has been poured into a gel tray and left to solidify
  • you need to put the gel tray into the gel box, making sure that the end of the gel tray with the wells is closest to the negative electrode on the gel box
  • add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in it
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12
Q

in the second step of electrophoresis, how are the samples loaded into the wells

A
  • using a micropipette, add the same volume of loading dye into each fragmented DNA sample (loading dye helps the samples to sink to the bottom of the wells and makes them easier to see)
  • add a set volume of a DNA sample into the first well (be REALLY careful when adding the samples, making sure the tip of the micropipette is in the buffer solution and just above the opening of the well - not TOO far down as it will pierce the bottom)
  • repeat the process and add the same volume of other DNA samples into the other wells (clean micropipette each time)
  • keep note of which sample is in each well
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13
Q

how is the third step of electrophoresis, where it actually occurs, carried out

A

1) lid is placed on the gel box and leads are connected from the box to power supply
2) power supply is turned on and set to particular voltage, causing an electrical current to pass through the gel
3) DNA fragments are negatively charged, so they’ll move through the gel towards the positive electrode (anode) at the far end
4) small DNA fragments move faster and further, causing them to all separate via size
5) let the gel run for 30 minutes, or until gel is about 2cm from the end, and turn off power
6) remove gel tray from the tank and tip off any excess buffer solution
7) wear gloves and stain the DNA fragments by covering the surface of the gel with staining solution and then rinse with water
8) will now be able to see visible bands of DNA fragments
9) the size of the DNA fragments are measured in bases (1000 = 1 kb kilobase)

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14
Q

what is an alternative method to dyeing the gel in electrophoresis

A
  • southern blotting:
  • where the DNA fragments are separated from the gel onto a nylon membrane
  • radioactive/fluorescent DNA probes (have complimentary short DNA/RNA sequence) are attached to label each specific fragment, called hybridisation
  • excess probes washed off
  • placed onto X-ray film/ under UV light
  • dark bands revealed where fragments are
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15
Q

why is electrophoresis possible in DNA

A
  • DNA fragments are negatively charged
  • DNA fragments are of different sizes
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16
Q

what can you or can’t you undertake electrophoresis on

A
  • RNA and DNA as negatively charged
  • not proteins, as can be positively or negatively charged
  • so before undergoing electrophoresis, they need to be mixed with chemical that denatures positive charge (so all equal charge)
  • many uses of this, e.g. in diagnosing diseases with urine and blood samples
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17
Q

how can you cut out DNA fragments

A

using restriction enzymes

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18
Q

what is a palindromic sequence

A
  • a sequence of nucleotides that consist of antiparallel base pairs (read the same in the opposite direction)
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19
Q

what are restriction enzymes

A

enzymes that recognise specific palindromic DNA base sequences (recognition sequences) and cut (digest) the DNA at these places

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20
Q

explain how each restriction enzyme differs

A
  • different restriction enzymes cut at different specific recognition sequences
  • as the shape of the sequence is complementary to the enzymes active site
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21
Q

how do restriction enzymes work

A
  • if recognition sequences are present at either side of the DNA fragment
  • the enzyme will separate it from the rest
  • needs to be incubated
  • and will cut the DNA fragment out via a hydrolysis reaction
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22
Q

what are sticky ends and how do they apply to restriction enzymes

A
  • small tails of unpaired bases at each end of the fragment
  • are useful as can be used to bind (anneal) the DNA fragment to another piece with the complementary sticky ends
  • as opposed to blunt ends
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23
Q

what is an organism’s genome

A

all of the genetic material that an organism contains

  • for us, all DNA in nucleus and mitochondria
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24
Q

what are non-coding base sequences called

A

introns
- removed from mRNA before it can be translated

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25
Q

what does our DNA contain

A
  • some coding genes, exons
  • loads of non-coding genes
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26
Q

what bit of the DNA is DNA profiling interested in

A
  • the repetitive, non-coding base sequences present in us
  • called satellite DNA, made of VNTRs and STRs
  • satellites will always appear in the same position on a person’s chromosome, but the number of repeats and length of each satellite will differ
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27
Q

why can DNA profiling be carried out

A
  • the number of times the non-coding sequences are repeated differs from person to person
  • so the length of these nucleotides differs too
  • because you inherit different lengths of repeats from your parents (identical twins the same, but closer you are related to someone, also the closer it will show)
  • can analyse the number of times a sequence is repeated at different, specific places (loci) in a person’s genome
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28
Q

what are the steps in DNA profiling

A

1) extraction of DNA: from a tissue sample, you DON’T need a lot, as can use PCR to amplify the amount that you have (primers bind to either side of the repeat so it is wholly repeated)
2) need to digest the sample: using restriction enzymes to cut as the specific points on introns
3) need to separate out the DNA fragments using electrophoresis to be analysed, using southern blotting and hybridisation
4) creates a DNA profile which can be analysed

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29
Q

what is the probability of 2 individuals having the same DNA profile

A
  • very low
  • as the chances of 2 individuals having the same number of sequence repeats at each locus in DNA is very low
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30
Q

what are the 2 main uses of DNA profiling

A
  • forensic science
  • medical diagnosis
  • species analysis
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31
Q

how is DNA profiling used in forensic science

A
  • compare samples of DNA collected at the crime scene, e.g. from blood, hair, semen, skin cells, hair to possible suspects, and link them
  • carry out making a DNA profile
  • and compare to see if any match the sample found at the crime scene
  • through having same pattern of bands in the gel (if matched, linked to crime scene)
  • same process also used to see if 2 things are the same species
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32
Q

how is DNA profiling carried out in medical diagnosing

A
  • DNA profiling could refer to a specific pattern of several alleles, or just non-coding repeats which are associated to a certain disease
  • analysing the risk of genetic disorders
  • useful especially where the specific mutation isn’t known, or where several mutations could have caused the disorder, as identifies a broader altered genetic pattern
  • e.g. is genetic diseases where you match up faulty regions in parent’s DNA to child’s DNA, and see if these have been inherited or not
  • also for immigration cases, proving paternity, evolutionary relationships
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33
Q

by which method can DNA be sequenced

A

chain-termination method

  • used to determine the order of bases in DNA
34
Q

what was the first method developed for sequencing DNA

A

Sanger sequencing

35
Q

what is added in tubes to begin DNA sequencing

A

4 different tubes:
1) single stranded DNA template - the DNA to sequence
2) lots of primer DNA - short pieces of DNA
3) DNA polymerase - enzyme that joins DNA nucleotides together
4) free nucleotides - lots of free ATCG
5) fluorescently labelled modified nucleotides (terminator bases) - similar to normal nucleotides, but once these are added to the strand, no more bases can be added after, in each tube, a different modified base is added, e.g. A/T…

36
Q

what is the first step in DNA sequencing, once everything has been added into the 4 different test tubes

A
  • the tubes are placed in a thermal cycler, and undergo PCR
  • this produces many strands of DNA
  • all will be different lengths, because they would have been terminated at different points depending on where the modified nucleotides have been added
37
Q

roughly explain how terminator bases help with dna sequencing

A
  • they are in much smaller volumes than normal bases
  • and will add at random points wherever their complementary base is present in the template strand, instead of the normal base
  • this will stop any more bases from adding, and terminate the strand
  • after a few cycles, a terminator base will have attached to all of the points possible on different strands
38
Q

what happens in DNA sequencing once PCR has occurred

A
  • the DNA fragments in each tube will undergo gel electrophoresis
  • special type in minute capillary tubes
  • will be visualised under UV light due to the fluorescent labels
  • the complementary base sequence can be read from the gel, with the smallest nucleotide at the bottom of the gel
  • each band after is one more base added
39
Q

which way do you read the results of the DNA sequencing

A
  • from bottom to the top
  • gives complementary sequence, so just switch back to give to give the original
40
Q

why has gene sequencing progressed so rapidly

A
  • continued research
  • advancements in modern technology
  • so can now sequence whole genomes more quickly
41
Q

how has the chain-termination technique advanced over time

A
  • become more automated and faster
  • the tubes contain all of the modified nucleotides, just with a differently coloured fluorescent label, which the machine reads for you
  • so you don’t have to run a gel and determine the bases, you get a computer read-out
42
Q

what is a new technique in DNA sequencing, and what are the pros

A

high-throughput sequencing
- sequences a lot faster than the original method, 1000 more bases in a given time
- at a fraction of the cost

43
Q

what is a specific method of high-throughput sequencing

A

high-throughput pyrosequencing
- doesn’t require use of electrophoresis

44
Q

explain pyrosequencing

A
45
Q

how does sequencing genes aid in predicting the sequences of amino acids in polypeptides

A
  • amino acids are coded for by a triplet of bases in a gene
  • so if you sequence a gene
  • you can predict the order of amino acids coded for by the gene, and therefore the primary structure of it
46
Q

what is synthetic biology and why has it emerged

A
  • once we know which genes code for which proteins, we can build biological molecules from scratch
  • a developing area of creating DNA from scratch, as opposed to genetic engineering, which is directly transferring DNA from one organism into another
47
Q

what are some of the applications of synthetic biology

A

1) building biological systems from artificially made molecules, to see whether they work in the way we think they do
2) redesigning biological systems to perform better and include new molecules (replacing faulty genes)
3) designing new biological systems and molecules that don’t exist in the natural world, but could be useful to us: fuels, drugs, e.g. artemisinin as an antimalarial drug, which we have now produced a precursor for, and inserted into yeast to produce

48
Q

what is computational biology

A

the use of computers to study biology, e.g. creating computer simulations and mathematical models

49
Q

what is bioinformatics

A

developing and using computer software that can analyse, organise and store biological data

50
Q

what has bioinformatics and computational biology led to the development of using DNA sequencing

A

genome-wide comparisons between individuals and between species

51
Q

how can sequenced genes help to study genotype-phenotype relationships

A
  • we can predict an organism’s phenotype by analysing its genotype
  • e.g. if a disease causes certain symptoms depending on its position and nature, this can be found out
  • you can sequence the gene of many people with disease, and make notes of their phenotype along the way
  • using bioinformatics, scientists can compare all the data, and find phenotype-genotype correlations
  • could help treating them, as gene sequencing can predict which problems they are likely to face
52
Q

what is epidemiological studies

A
  • epidemiology is the study of health and disease within a population
  • considering the distribution of the disease, its causes and effects
  • some gene mutations have been linked to a greater risk of the disease
  • computerised comparisons between genomes of people with and without the gene can detect the particular mutation responsible for the increased risk of disease
53
Q

how can comparing genomes help us understand evolutionary relationships

A
  • all organisms evolved from shared common ancestors
  • closely related species evolved away from each other more recently
  • and share more DNA
  • whole genomes of species can be sequenced and analysed using computer software to tell us how closely related two species are
  • comparing genomes of members of the same species can also tell us about evolutionary relationships
  • when different groups of early humans separated and moved to different parts of the world, their genome changed slightly in different ways
  • using a computer to compare people from different parts of the world
  • helps to picture early human migration
54
Q

what are genetically engineered organisms

A
  • organisms that have had their DNA altered (manipulated) by genetic engineering
  • also called transformed organisms
55
Q

what DNA do GMO have and what is this

A

recombinant dna:
- dna formed by joining together dna from different sources

56
Q

what is the basic overview of genetic engineering

A
  • extracting a gene from one organism
  • and inserting it into another organism, usually one of another species
  • genes can also be manufactured, instead of extracted
  • organism with the inserted gene will produce the protein coded for by that gene
57
Q

what is a transgenic organism

A

an organism that has been genetically engineered to include a gene from a different species

58
Q

what is the first step in genetic engineering

A

1) obtain the DNA fragment containing the gene you want
- usually using a restriction enzyme (e.g. restriction endonuclease)
- could also be from mRNA, forming cDNA

59
Q

what is the second step in genetic engineering, once the desired gene has been obtained

A
  • the isolated DNA fragment is then inserted into a vector using restriction enzyme and DNA ligase
60
Q

what is a vector

A

something that’s used to transfer DNA into a cell
- e.g. plasmids (small, circular molecules of DNA in bacteria)
- e.g. bacteriophages ( viruses that infect bacteria)

61
Q

how is the vector prepared before the desired DNA can be inserted

A
  • the vector DNA is cut at the same restriction enzyme that was used to isolate the DNA containing the desired gene
  • so the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
62
Q

what is the point of DNA ligase in the second step of genetic engineering

A
  • the vector DNA and DNA fragments are mixed together using DNA ligase
  • which joins the sugar-phosphate backbone of the two bits
  • process called ligation
  • new combination of DNAs is called recombinant DNA
63
Q

what is the third step of genetic engineering

A
  • the vector is used to transfer the gene into the bacterial cell
64
Q

how can the recombinant DNA, if on a plasmid vector, be reintroduced into a bacterial cell

A
  • through electroporation:
  • where the bacterial cells are mixed with the plasmid vector and placed in an electroporator
  • the machine is switched on
  • and electrical field is created in the mixture
  • increased the permeability of the bacterial cell membrane
  • allows it to take in the plasmid
65
Q

how would a bacteriophage vector take up the gene

A
  • bacteriophage will infect the bacterium y injecting its DNA into it
  • the phage DNA with desired gene then integrates into the bacterial DNA
66
Q

why are the plasmids chosen often containing a marker gene

A
  • makes it easy to see which bacteria have actually taken up the plasmid
  • e.g. for antibiotic resistance, if have the plasmid, will grow anyways on agar with disease, as taken up the plasmid containing this marker gene
67
Q

what is the point of a second marker gene

A
  • to show that the plasmid contains the recombinant gene
  • often, the marker gene is inserted into the plasmid itself by genetic engineering
  • and the plasmid is cut up here and the desired gene inserted, meaning the second marker gene doesn’t express itself
  • so if the gene is not expressed, shows that the plasmid contains recombinant DNA
68
Q

GENETICALLY MODIFIED ORGANISMS CARDS- NOT COVERED PLEASE DO

A
69
Q

what are genetic disorders

A

inherited disorders caused by abnormal genes or chromosomes

70
Q

what does gene therapy involve

A

altering alleles inside cells to cure genetic disorders

71
Q

what is the process behind how you do gene therapy dependant on

A

whether the genetic disorder is caused by two recessive or one dominant allele

72
Q

how would gene therapy work for a recessive disorder

A

by adding a working dominant allele to make up for them

73
Q

how would gene therapy work for a dominant disorder

A

you have to “silence” the dominant allele, e.g. by adding a piece of DNA in the middle to the allele so it doesn’t work anymore

74
Q

how do you get the new DNA inside the cell in gene therapy

A
  • allele is inserted into the cell using vectors
    e.g. viruses, plasmids, liposomes (spheres made of a lipid)
75
Q

what are the 2 types of gene therapy

A

somatic and germ line

76
Q

what is somatic therapy

A
  • involves altering the alleles in body cells, particularly the cells that have been most affected by the disorder
  • doesn’t affect an individual’s sex cells, so offspring can still inherit the disease
    e.g. cystic fibrosis: which is affected most in the respiratory system, so the somatic therapy for this targets the epithelial cells lining the lungs
77
Q

what is germ line therapy

A
  • involves altering the alleles in sex cells
  • means that every cell of any offspring will be affected by the gene therapy and would not inherit the disease
  • currently illegal in humans tho
78
Q

what are the positive ethical issues of gene therapy

A
  • prolong life of people with genetic disorders
  • give people with genetic disorders a better quality of life
  • carriers of disorder might be able to conceive a baby without that disorder or risk of cancer (only germ line)
  • decrease the number of people suffering from genetic disorder (only germ line)
79
Q

what are negative ethical issues with gene therapy

A
  • technology at risk of being used for things other than medical disorders, e.g. cosmetic effects of aging
  • potential of doing more harm than good by using the technology, e.g. overexpression of genes
  • concern of it being expensive, health service resources should be better well spent on other treatments that have actually passed clinical trials
80
Q

what are other disadvantages of gene therapy

A
  • may only be short-termed effect (somatic)
  • patients may have to undergo multiple treatments (somatic)
  • might be difficult to get the allele into specific body cells
  • body could identify the vectors as foreign bodies and start an immune response
  • allele could insert into the wrong part of the DNA, causing more problems, e.g. cancer
  • an inserted allele might get overexpressed, producing too much of the missing protein

Biology Module 6 (6 decks)

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