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Bio > Manipulating DNA - Unit 3 AOS 1 > Flashcards

Manipulating DNA - Unit 3 AOS 1 Flashcards

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Biology 101 flashcards
1
Q

endonucleases (restriction enzymes)

A
  • an enzyme that breaks phosphodiester bonds between two nucleotides in a polypeptide chain
  • they cut the sugar-phosphate backbone at specific recognition sites
  • they naturally occur in bacteria and can act as a defence mechanism against invading bacteria
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2
Q

sticky end

A
  • the result of a staggered cut through DNA
  • overhanging nucleotides
  • they are sticky because they will be attracted to unpaired complementary nucleotides
  • advantageous becuse DNA will be oriented the righ way
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3
Q

blunt end

A
  • the result of a straight cut across DNA, resulting in no overhanging nucleotides
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4
Q

Ligases

A
  • enzymes that join two pieces of DNA at their sugar-phosphate backbone, catalysing the formation of a phosphodiester bond
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5
Q

polymerases

A
  • DNA polymerase synthesis DNA molecules from nucleotides
  • RNA polymerase synthesises RNA molecules from a strand of DNA
  • they require a primer
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6
Q

Primer

A
  • a short stand of nucleic acids which acts as a starting point for polymerase enzymes to attach to.
  • they are complementary to the template strand
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7
Q

CRISPR Cas9

A
  • a complex formed between Cas 9 and gRNA which is found in bacteria
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8
Q

Cas 9

A
  • an endonuclease that cuts DNAat a point determined by gRNA
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9
Q

gRNA

A
  • a specific sequence of RNA which recognises the desired DNA and directs the Cas 9 to cut the DNA
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10
Q

protospacer

A
  • a short sequence
    of DNA extracted from a
    bacteriophage by Cas1 and Cas2,
    which has yet to be incorporated
    into the CRISPR gene
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11
Q

Spacer

A
  • short sequences of
    DNA obtained from invading
    bacteriophages that are added into
    the CRISPR sequence
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12
Q

protospacer adjacent motif (PAM)

A
  • a sequence of two-six nucleotides
    that is found immediately next to
    the DNA targeted by Cas9
  • cas 1 and cas 2 search for a PAM sequence and cut out a photospacer just before it so the PAM sequence is not included in the photospacer.
  • cas 9 also searches for a pam sequence next to the targen DNA so that is doesn’t need to search through every sequence of DNA
  • PAM sequences are never found in bacterias own CRISPR repeats to that Ca9 9 verver cuts its own DNA
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13
Q

CRISPR Cas 9 defence system

A
  • exposure - the bateriophage injects some of its DNA into the bacteria. CAS 1 and CAS 2 cut out a protospacer which is introduced to the genome to become a spacer
  • expression - the CRISPR spacers are transcribed and converted into gRNA. gRNA binds directly to Cas 9 creating a CRISPR cas9 complex. This is directed to any viral DNA that is complementary to the gRNA
  • extermination - Cas 9 cuts the sugar phosphate backbone inactivating the virus
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14
Q

repair mechanisms

A
  • when the viral DNA is cut, the bacterium will want to repair it
  • mutations in the repair mechanisms will inactivate the virus
  • the whole process will continue until a mutation occurs to inactivate the virus.
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15
Q

genetic modifications

A
  • the manipulation of an organism’s genetic material using biotechnology
  • removing or replacing DNA within a genome
  • it can be used to fix deleterious mutations which negatively affect the organism
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16
Q

gene therapy

A
  • repairing genetic mutations by replacing a defective gene with a healthy one
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17
Q

sgRNA

A
  • guide RNA utilised by scientists to instruct Cas9 to cut a specific site
    when using CRISPR-Cas9 in gene editing
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18
Q

using CRISPR cas 9 in gene editing

A
  • Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut.
  • A Cas9 enzyme is obtained with an appropriate target PAM sequence
  • Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR Cas 9 complex
  • The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.
  • The Cas 9 finds the target PAM sequence and checks whether the sgRNA aligns with
    the DNA.
  • Cas9 cuts the selected sequence of DNA
  • The DNA has a blunt end cut that the cell will attempt to repair.
    8 When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site.
  • Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap.
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19
Q

gene knockout

A
  •  a technique in gene editing where scientists prevent the expression of a target gene to understand its function in an organism
20
Q

gene knockin

A
  • a technique in gene editing where scientists substitute or add nucleotides in a gene
21
Q

ethical implications to CRISPR Cas 9

A
  • scientists must treat an embryo prior to it differentiating
  • scientists cannot get consent from embryos to alter their genes
  • ## equity of access
22
Q

polymerase chain reaction

A
  • an artificial method of amplifying DNA in lab conditions
  • it replicated DNA by making multiple identical copies.
  • it is used by scientists when there is an insufficient amount of DNA
23
Q

steps of the polymerase chain reaction

A
  • denaturing - the DNA is heated to approximately 95 degrees so that the hydrogen bonds between base pairs will break, separating the two stands
  • annealing - the DNA is heated to approximately 55 degrees allowing primers to bind to complementary sections of the single stands of DNA.
  • elongation - the DNA is heated back up to 72 degrees which is the optimal temperature of taq polymerase, allowing it to bind to the primers and begin synthesising two new stands of DNA
    -the process is then repeated until enough DNA has been amplified.
24
Q

taq polymerase

A
  • a heat resistant DNA polymerase isolated from the bacteria thermus aquaticus
  • it binds to primers during the annealing stage and begins synthesising a new complementary stand of DNA
  • it is used instead of human DNA polymerase because it can withstand high heat without denaturing
25
Q

primers

A
  • primers are used to bind to the stands of DNA allowing taq polymerase to function
  • a forward primer binds to the 3 prime end of the template stand and synthesises a new stand in the same direction as RNA polymerase would function
  • a reverse primer binds to the 3 prime end of the coding stand, synthesising a new stand of DNA in reverse.
26
Q

gel electrophoresis

A
  • a technique used to measure the length of DNA, based on molecular size as well as how many different sizes of DNA fragments are in a sample
  • smaller fragments move further because they can weave through the gel easier.
  • DNA starts at the negative end and moves towards the positive end because DNA has a negative phosphate group
27
Q

agarose gel

A
  • a gel that contains pores for the DNA to move through
28
Q

buffer

A
  • a solution containing ions which carries an electrical current through the gel
29
Q

standard ladder

A
  • a mixture of DNA fragments of known length that are used as a comparison
30
Q

controlled variables in gel electrophoresis

A
  • voltage of the power source
  • percentage of agarose in the gel (agarose is a polysaccharide added to the gel to control
    the rate at which fragments move through the gel)
  • concentration of the solution (a ‘buffer’) covering the agarose gel
  • temperature of the environment
  • pH of the buffer solution
  • length of time the sample of DNA is allowed to run for in the gel
  • length of the gel (distance between the negative and positive electrodes).
31
Q

factors impacting the movement of DNA

A
  • voltage - the higher the voltage, the faster the DNA will move
  • gel composition - gels with greater density and agarose concentration will be harder for DNA to move through
  • buffer concentration - the greater the concentration of ions in the buffer, the greater the electrical current
  • time - the longer the DNA is left, the more it will travel
32
Q

DNA profiling

A
  • a method of DNA analysis where regions of DNA from different individuals are analysed and compared
  • coding regions of DNA are not useful because they code for gene products that everybody has
  • STRs are small sections of repeated
    nucleotides that vary in length between people and are found in the non-coding areas
    of autosomal chromosomes
  • STRs can vary significantly between individuals
  • several variable number tandem repeats are studies so that there is a very low chance of two individuals having the exact same number of nucleotide repeats.
33
Q

heterozygous and homozygous in DNA profiling

A
  • if an individual is heterozygous for an STR, they will have two bands
  • if an individual is homozygous for an STR, they will have one thicker band
34
Q

making a recombinant plasmid

A
  • the gene that scientists want to be expressed in recombinant plasmids (gene of interest) will be inserted into a vector
  • it does not have introns
35
Q

plasmid vector

A
  • a piece of circular DNA that is modified to be an idea vector for bacteria transformation
  • i site on the plasmid can be recognised and cut by a restriction endonuclease
  • DNA ligase is then needed to join the sugar phosphate backbone
36
Q

transforming bacteria

A
  • recombinant plasmids are inserted into the cytoplasm of bacteria (bacterial transformations
  • heat shock involves rapidly increasing and decreasing the temperature to increase membrane permeability
  • electroporation involves delivering electric shocks to bacteria membranes to increase its permeability
37
Q

antibiotic selection

A
  • to determine between transformed and untransformed bacteria, the mixture is cultured on an agar plate
  • the untransformed bacteria will be killed off because it doesn’t contain the gene necessary for antibiotic resistance.
38
Q

making insulin - making the recombinant plasmids

A
  • plasmid vectors prepared which contain the amp gene and tet gene to code for antibiotic resistance. tet gene has a recognition site to one of the restriction endonucleases
  • two plasmids are used (A and B). EcorI and BamHI are used to cut the insulin genes and the plasmids, creating sticky ends. This means that the insulin should be complementary to the cut space in the vector and the two restriction enzymes means the insulin will co in the correct orientation
  • DNA ligase completes the sugar phosphate backbone, creating recombinant plasmids.
39
Q

making insulin - bacterial transformation

A
  • the plasmids are mixed with E.coli
  • to determine which bacteria has taken up plasmids, they are grown on a plate containing ampicillin. the bacteria that grows, has a plasmid
  • to determine which ones are recombinant, they are grown on a plate containing tetracycline. the ones that don’t grow, contain recombinant plasmids.
40
Q

genetic engineering

A
  • the process of using biotechnology to alter the genome of an organism
41
Q

genetically modified organism

A
  • an organism that has had its genome altered through genetic engineering technology
42
Q

cisgenic organism

A
  • a genetically modified organism that has DNA from the same species inserted into it
43
Q

transgenic organism

A
  • a genetically modified organism that has material from a different species inserted into its genome (recombination)
44
Q

GMOs in agriculture

A
  1. a gene of interest with desirable characteristics is isolated - either synthetically or it is taken from an organism using restriction endonucleases
  2. the gene is amplifies using PCR and inserted into a plasmid vector and then inserted into e recipient organism
  3. the transformed and recombinant plasmids are identified using antibiotic resistant genes or fluorescent genes.
  4. the transformed cells are then grown into GMO organisms
45
Q

benefits of GMO in agriculture

A
  • increased crop productivity: improving the crop yield and the quality of crops (nutritional value and ability to grow in certain conditions
  • increased disease resistant - genes can be inserted into crops that make them less impacted by harmful pathogens
46
Q

Gel electrophoresis steps

A
  • samples containing DNA fragments and a die are loaded into wells at the negative end to the gel.
  • the gel is immersed into a buffer solution containing ions so an electric charge can pass through
  • a current passes through the gel using two electrodes. The DNA moves from the positive end to the negative end.
  • smaller fragments move further
  • at the end, a different dye that attaches to DNA is used so that the bands can become visible.

Bio (22 decks)

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