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VCE BIOLOGY U3/4 > CHAPTER 2: DNA MANIPULATION > Flashcards

CHAPTER 2: DNA MANIPULATION Flashcards

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1
Q

DNA polymerase

A

uses DNA polymerase to accurately copy a DNA template

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2
Q

endonucleases - restriction enzymes

A
  • restriction enzymes like endonucleases cut DNA at specific recognition sequences known as restriction sites, splitting DNA into smaller fragments
    • restriction sites is a particular order of nucleotides
  • endonucleases make one incision on each of the 2 complementary strands of DNA
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3
Q

sticky ends

A
  • endonucleases cut one strand at one point but cut the second strand at a point that is not directly opposite → causes an overhang to form
  • DNA ligase enzyme connects the single-stranded DNA together via the sugar-phosphate back bones
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4
Q

which pairs faster, sticky or blunt

A
  • pieces of DNA with sticky ends pair faster than pieces of DNA with blunt ends
    • sticky ends allow for complementary base pairing so the pieces are held together by weak hydrogen bonds and can be acted on by the DNA ligase enzyme.
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5
Q

blunt ends

A
  • endonuclease cuts the 2 strands of DNA molecule at points DIRECTLY OPPOSITE each other to produce cut end
  • DNA fragments are joined directly together through the use of DNA ligase
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6
Q

DNA ligase

A
  • an enzyme known as ligases catalyses the joining of pieces of double-stranded DNA at their sugar-phosphate bones
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7
Q

what are plasmids

A
  • circular, double stranded DNA that can reproduce independently
    • can be taken up by bacterial cells
    • many are used as vectors to transport foreign DNA into bacterial cells to transform them
    • they have multiple recognition sites (restriction sites) for endonucleases
      • can have new genes inserted
  • have an antibiotic resistant marker (selective marker)
  • have a promoter or origin point for self replication
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8
Q

making recombinant plasmids

A
  • cut plasmid DNA with endonuclease
  • cut foreign DNA with the same endonuclease
  • mix plasmids and foreign DNA
  • add DNA ligase which joins the sticky ends together
  • same recognition site allows same restriction enzyme to cut both the plasmid and the gene of interest
  • produces matching sticky ends on the plasmids and the gene of interest
  • plasmids will then join with the DNA of the gene of interest to form recombinant plasmids as they have complementary sticky ends.
  • the two fragments can then be successfully joined by DNA ligase.
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9
Q

recombinant plasmid

A

a plasmid that has taken in new DNA is called a recombinant plasmid

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10
Q

bacterial transformation

A
  • when bacteria cells take up plasmids
  • recombinant plasmids need to be taken up by bacteria
  • techniques that temporarily interfere with the plasma membrane allow this (causes holes in the pm so plasmid can enter)
    • electroporation
    • heat shock
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11
Q

how is transformed bacteria identified

A
  • bacteria can take up recombinant plasmid with AmpR gene
    • results in bacteria resistant to ampicillin antibiotic
  • bacteria can take up recombinant plasmid with green fluorescent protein (gfp gene_
    • when uv light is used, bacteria is fluorescent
    • but needs arabinose , sugar to repress the repressor of the gfp gene
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12
Q

ethical issues of gene cloning

A
  • changing a species’ DNA may result in unforeseen consequences
  • concern that the pharmaceutical product may contain bacteria that will cause disease
  • not natural and therefore may be against religious or moral views
  • respect → embryos cannot give informed consent
  • respect → some believe that modifying embryos does not honour the sanctity of life
  • justice → gene editing technology is expensive and may only be available to wealthy people
  • non-maleficence → treated individuals or embryos may experience unforeseen side effects
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13
Q

purpose of LB broth

A
  • provides nutrients to support bacterial growth
  • both transformed and untransformed bacteria can grow
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14
Q

purpose of ampicillin

A
  • to identify which bacteria has been transformed
  • bacterial growth → has the +pREC because it has AmpR gene, resistant to ampicillin antibiotic
  • no bacterial growth → is -pREC no AmpR gene
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15
Q

purpose of arabinose

A
  • to also identify which bacteria has been transformed
  • arabinose represses the repressor of the GFP gene (binds to protein AraC)
    • allow the GFP gene to be expressed
    • so under UV light, bacteria will appear green
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16
Q

potential errors
- transforming bacteria experiment

A
  • bacteria was left in the heat bath for too long, killing the bacteria before plating it
  • the wrong test tube was put on the plate
  • no recomb test tube was added to the wrong plate (plates mixed up) → human error
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17
Q

benefits of recombinant insulin

A
  • high levels of purity
  • reiability of supply
  • reduced chance of side effects such as allergies (compared to pig or cow derived insulin)
  • consistency of quality between batches
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18
Q

gel electrophoresis

A
  • a method used to separate DNA fragments based on size (length, measured in bp)
  • the phosphate group of nucleotides is negatively charged
    • negatively charged DNA moved towards the positive terminal (b/c it is negatively charged)
  • DNA is loaded into a gel that acts like a sieve to allow SMALLER DNA fragments through more quickly than larger ones
  • the result of gel electrophoresis is a series of PARALLEL BANDS of DNA fragments at differing distances down the gel
    • each band can contain millions of DNA molecules of the same size
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19
Q

applications of gel electrophoresis

A
  • people have DIFFERENT DNA SEQUENCES, so when endonucleases are used, the DNA will be cut at DIFFERENT LOCATIONS and DNA fragments of different lengths will result
  • gel electrophoresis can identify people in:
    • forensic investigations
    • mass disasters
    • paternity testing
    • identifying animals
20
Q

mitochondrial DNA

A
  • not all DNA is used in gel electrophoresis
    • nuclear or mitochondrial DNA can be used
  • compared to nuclear DNA, mtDNA
    • is inherited via the MATERNAL LINE
    • does not recombine during reproduction → LESS VARIABLE
    • is present in LARGER AMOUNTS
21
Q

dna profiling - short tandem repeats

A
  • chromosomal sites (non coding region) where many copies of a short DNA sequence are joined end-to-end; the number of repeats is variable between unrelated people
  • STRs are 2-5 bp in length and repeated over and over
  • the number of REPEATS VARIES between people and each variation is a distinct allele
  • we have 2 copies of each STR - one from mother and one from father
22
Q

types of DNA used for DNA profiles

A
  • to develop DNA fingerprints or DNA profiles, regions with high variation between individuals are used.
    • Short Tandem Repeats (STRs) from nuclear DNA
      • STRs can identify individuals
    • Hypervariable regions (HVRs) in mtDNA
      • mtDNA can identify relationships/when fewer cells are available
23
Q

bacteriophages

A
  • there are viruses called bacteriophages
    • they inject viral DNA into the bacteria
    • they hijack the bacteria’s cellular machinery to reproduce to make more bacteriophages
    • this causes the bacteriophage to burst out and cycle repeats to infect more bacteria
24
Q

CRISPR regions

A
  • CRISPR is an immune response found naturally in bacteria and is modified in the lab to edit genes
  • CRISPR provides a form of NATURAL IMMUNITY for the bacteria against viral attacks
  • DNA with short repeated sequences (repeats) and spacers
    • CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS
  • each repeat is separated by a ‘spacer’ DNA
    • the spacer DNA is a segment of DNA from bacteriophages the cell has encountered previously
  • these clusters are regularly interspaced with the repeat DNA
25
Q

spacers

A
  • are made up of remnants of DNA from bacteriophages of foreign DNA
    • when bacteria encounters a new virus, they store some of its DNA in a new spacer sequence
26
Q

how does CRISPR-Cas9 work

A
  • a bacteriophage attaches to the outside of bacterial cell and injects its VIRAL DNA into the cell (reinfection)
    • previously, a segment of the viral DNA has been stored as a spacer in the CRISPR region
  • the CRISPR sequence is TRANSCRIBED resulting in pre-CRISPR RNA (crRNA)
  • tracer RNA (trcrRNA) has a complementary sequence to the repeat DNA (NOT the spacers)
    • role: helps hold the gRNA in place in the Cas9 enzyme
  • RNAase cuts spacer, portion of the repeat and associated tracr-RNA to become g-RNA
  • the specific spacer of the crRNA binds to the trcrRNA to form guide RNA (gRNA)
  • gRNA then binds with the Cas9 enzyme → forms a Cas9-gRNA complex
  • Cas9-gRNA complex scans bacteriophage (target DNA) to look for complementary bases
  • once it is found, the DNA is unzipped and Cas9 cuts/cleaves the DNA creating blunt DNA fragments.
  • DOUBLE BLUNT END CUT
  • the viral DNA cannot reproduce as the DNA has been disrupted
27
Q

uses of CRISPR Cas9

A
  • scientists can program CRISPR-Cas9 system to TARGET ANY SEQUENCE using the gRNA
  • can introduce a sgRNA (single guide RNA) that is complementary to the gene of interest
  • knock out genes one at a time, in order to identify their function
  • to introduce specific mutations in a DNA sequence
  • to edit a faulty allele of a gene in a person with a severe inherited disease
  • to snip out the faulty segment of a gene and replace it with a working copy
  • to activate or to repress a gene
  • to add a new gene to the genome

it is an easy and cost-effective way of editing an organism’s genome

28
Q

what is PCR used for

A
  • used to amplify (make more copies) of a segment of DNA
  • process depends on the enzyme DNA polymerase → specifically Taq polymerase
  • doubles the amount of DNA with EACH CYCLE
29
Q

steps in PCR

A
  • denature
    • the DNA sample is heated 94 degrees to 98 degrees for one minute
    • this breaks the hydrogen bonds between the double strands
    • separates DNA into two single, separate strands
  • anneal
    • PRIMERS are added
      • short segments of SINGLE STRANDED DNA
    • primers bind to the target DNA (at the complementary sequences), initiating DNA synthesis 50 degrees to 65 degrees for two minutes
  • extension
    • taq polymerase starts at the primers and extends them using free nucleotides
      • forms TWO COMPLETE DOUBLE STRANDS
      • occurs at around 72 degrees for one minute → optimal temperature of taq polymerase
  • the process is then repeated
30
Q

ethical requirements of human experimentation

A
  • Respect for persons: Informed consent
    • E.g. people must know what they are agreeing to (harms, likelihood of success, etc.)
  • Integrity: Being honest and trustworthy
    • E.g. Being open about the likelihood of benefit or harm
  • Justice: Selection of human subjects and non-discrimination
    • E.g. vulnerable people not targeted
  • Beneficence: Maximising benefit and minimizing harm
    • E.g. the welfare of the person is most important (the research comes second)
  • Non-maleficence – do no harm
    • Eg. Stopping a treatment if it is causing harm
31
Q

CRISPR Cas9 ethical considerations

A
  • long-term impacts of the technology and informed consent risks
  • possibility of the edited genes being inherited and affecting future generations
  • the use in individuals who may have not provided consent
  • misusing technology such as around the use for designer babies
  • confidentiality considerations of genetic data
  • personal beliefs and opinions around the editing of human DNA (such as religious or cultural objections).
32
Q

how to run a gel electrophoresis

A
  1. DNA sample is combined with DNA loading dye.
  2. the mixture is placed in a well at one end of the agarose gel.
  3. the agarose gel is immersed in a buffer solution (salt solution).
  4. it is then exposed to an electric field with the positive(+)pole at the far end and the negative(−) pole (cathode) at the well
  5. smaller fragments move through the agarose gel faster compared to larger fragments.
  6. these fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.
33
Q

how can CRISPR Cas9 be programmed to cut any DNA sequence

A
  • scientists create a complex, consisting of a gRNA sequence and the Cas9 protein
    • gRNA sequence is complementary to the target DNA sequence
  • Using the gRNA, Cas9 identifies the complementary DNA sequence and unwinds the DNA and cuts it
  • scientist can insert a new piece of DNA, remove, replace or add or delete single nucleotides at the site of the cut
  • the cell detects and repairs the broken strands of DNA
34
Q

purpose of the PAM sequence

A
  • a highly specific region
  • a very short nucleotide sequence adjacent to the target spacer (PAM sequence)
  • using gRNA, cas9 enzyme searches viral DNA for a PAM
  • upon finding it, CAS9 complex compares the sequence of bases in the crRNA to a sequence upstream of the PAM
  • If they are complementary, Cas9 cuts the DNA, preventing infection.
  • also where Cas9 binds to on the target DNA sequence
  • plays an essential role in distinguishing self bacterial DNA from non-self viral DNA
35
Q

purpose of Cas9 enzyme

A
  • Cas9 will only cut out the target sequence if it recognises a very short nucleotide sequence adjacent to the target spacer called a protospacer adjacent motif (PAM) sequence.
  • Cas9 is an endonuclease associated with CRISPR and acts likes a pair of molecular scissors capable of precisely cutting both strands of DNA.
36
Q

GMO definition

A

genetically modifies organisms are organisms that have had their DNA directly manipulated

37
Q

transgenic definition

A
  • transgenic organisms are a subset of GMOs that have DNA from different species
  • eg. transforming bacteria with recombinant plasmid
38
Q

how GMO can increase crop productivity

A
  • roundup ready canola
  • canola plants are used to make canola oil
  • roundup is a herbicide
  • roundup ready canola has a bacterial gene inserted
  • this allows the canola plant to make an enzyme to break down the herbicide roundup
  • GM canola crops can be sprayed with the herbicide and only the weeds die, leaves the GM canola crops unaffected
39
Q

how GMO can provide pest resistance

A
  • Bt cotton has a bacterial gene that allows it to produce chemicals that will kill insects
  • the crops are not eaten by insects → resulting in more cotton production
  • crops do not need to be sprayed with insecticide
40
Q

compare genetic modification to other methods

A
  • faster (traditional techniques like artificial selection took time to breed the organisms with the favourable traits)
  • more precise (edit specifically the DNA for the trait desired)
41
Q

what is more useful for gel electrophoresis- sticky or blunt ends

A
  • blunt ends
  • sticky ends may allow for the hydrogen bonds between the complementary base pairs to reform
  • causes fewer bands on the gel - less reliable results
42
Q

making synthetic insulin

A
  • human insulin is composed of 2 polypeptide chains
    - an A chain and a B chain. It is therefore a quaternary structure.
  • the A chain and the B chain are synthesised separately on separate plasmids in separate bacteria.
  • the plasmid used has antibiotic resistance selectable markers to allow for recombinant plasmid selection.
  • the insulin chain that is inserted into the plasmid has been modified to remove all introns as prokaryotes don’t have Introns.
  • the insulin genes are inserted next to a gene for β-galactosidase protein, which allows for detection of successful gene insertion.
  • The beta gal gene has 2 functions
    • its an additional selectable marker
    • the beta gal gene is an inducible operon and therefore can regulate A chain production
  • expression of each gene from the two bacteria allows a functional fusion protein to be produced.
  • once the genes are expressed and the fusion proteins are produced by each bacteria, these fusion proteins are then purified
  • the insulin polypeptides are removed and then combined together to produce functional insulin.
43
Q

difference between Cas9 and a typical endonuclease

A
  • Cas9 requires a PAM sequence adjacent to the target site in order to cleave
  • typical restriction enzymes cut at a set recognition site sequence, whereas Cas9 cuts at any target DNA sequence that is designated by single guide RNA (target DNA sequence is complementary to the sgRNA)
44
Q

difference between gRNA and sgRNA

A
  • gRNA (guide RNA) is comprised of 2 RNA molecules: crRNA and tracrRNA
  • scientists manufacture a synthetic sgRNA (single guide RNA) that is comprised of 1 RNA molecule
    sgRNA serves as both the tracrRNA and crRNA (containing the spacer)
45
Q

the role of primers and why we need to have two DIFFERENT primers

A
  • A primer attaches to complementary DNA nucleotides.
  • The addition of the primer and its joining to complementary nucleotides then allows the DNA polymerase to begin copying.
  • Two primers are needed as the nucleotide sequence is different at the start and finish of the section of DNA that must be copied.
46
Q

function of ORI on a plasmid

A

the site at which the plasmid replicates

47
Q

how can gel electrophoresis be used to determine if a plasmid is recombinant?

A
  • Adding foreign DNA to make a plasmid recombinant would increase its size.
  • when run through the gel electrophoresis you can then compare the banding pattern of the plasmid sample to the control sample and DNA ladder of known sizes
  • If you see additional bands in the plasmid sample that are not present in the control, these bands correspond to the insert DNA, indicating that the plasmid is recombinant.

VCE BIOLOGY U3/4 (13 decks)

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