CHAPTER 2: DNA MANIPULATION Flashcards

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1
Q

DNA polymerase

in DNA manipulation

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

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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8
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
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9
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
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10
Q

dna profiling - short tandem repeats

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
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11
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
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12
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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13
Q

making recombinant plasmids

A
  • cut plasmid DNA and 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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14
Q

recombinant plasmid

A

a plasmid that has taken in new/ foreign DNA

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15
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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16
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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17
Q

purpose of LB broth

A
  • provides nutrients to support bacterial growth
  • both transformed and untransformed bacteria can grow
18
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
19
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
20
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
21
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
22
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.
23
Q

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

A
  • sticky ends facilitate ligation by DNA ligase by forming hydrogen bonds between complementary bases of the other strand.
24
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
25
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
26
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
27
Q

how does CRISPR-Cas9 work

A
  1. 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
  2. the CRISPR sequence is TRANSCRIBED resulting in pre-CRISPR RNA (crRNA)
  3. 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
  4. RNAase cuts spacer, portion of the repeat and associated tracr-RNA to become g-RNA
  5. the specific spacer of the crRNA binds to the trcrRNA to form guide RNA (gRNA)
  6. gRNA then binds with the Cas9 enzyme → forms a Cas9-gRNA complex
  7. Cas9-gRNA complex scans bacteriophage (target DNA) to look for complementary bases - PAM SEQUENCE
  8. once it is found, the DNA is unzipped and Cas9 cuts/cleaves the DNA creating blunt DNA fragments.
  9. DOUBLE BLUNT END CUT
  10. the viral DNA cannot reproduce as the DNA has been disrupted
28
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
29
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
30
Q

denature

steps of PCR

A
  • 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
31
Q

anneal

steps of PCR

A
  • 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
32
Q

extension

steps of PCR

A
  • 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
33
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
34
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).
35
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
36
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
37
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.
38
Q

GMO definition

A

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

39
Q

transgenic definition

A
  • transgenic organisms are a subset of GMOs that have DNA from different species
  • eg. transforming bacteria with recombinant plasmid
40
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 that 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
41
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