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dna manipulation Flashcards

1
Q

what are endonucleases?

A

enzymes that can break the sugarphosphate bonds between nucleotides of nucleic acids
- cuts dna

scissors

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2
Q

what are restriction enzymes?

A

type of endonuclease produced by bacteria
- cut dna at their specific sequences/recognition site

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3
Q

why is it useful to cut with the same restriction enzymes?

A
  • will be specific to one sequence/has unique recognition sites
  • plasmid and target gene cut sites will have complementary nucleotides overhanging
  • attracted to each other= more likely target gene will insert in the plasmid
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4
Q

what are sticky ends?

A

a way in which dna is cut leaving overhanging unpaired nucleotides

exposed bases able to join to other dna with complementary sticky ends

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5
Q

what are blunt ends?

A

no overhanging nucleotides

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6
Q

what is dna ligase?

A

enzymes that join nucleic acid fragments together
- creates the bods between sugar and phosphate to join dna

glue/tape

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7
Q

what are polymerases

A

enzymes that join nucleotides together to create nucleic acids
- synthesises dna/amplifies fragments of dna

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8
Q

what is the function of crispr cas9 in bacteria

A

help them fight off viral infections
- target and cut viral dna= protect the cell

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9
Q

outline the process of how bacteria uses crispr

A
  • virus injects viral dna
  • viral dna added to bacterial genome
  • guide rna formed complementary to viral dna
  • grna guides cas9 to target gene and destroys viral genome
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10
Q

what is cas9 and its role

A
  • endonuclease that carries a piece of rna inside it
  • sequence of dna found is complementary to the guide rna
  • cas9 cuts
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11
Q

how can scientists use crispr cas9

A

programmable
- identify the desired dna sequence
- crispr cas9 can be directed to it

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12
Q

what is the role of sgrna/grna

A

guides cas9 and tells it where to cut

grna: bacteria. sgrna: made by scientists

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13
Q

how is cas9 programmed

A

changing sgrna
- identify desired sequence
- create sgrna made complementary to that target gene
- sgrna joined to cas9
- = cas9 now programmed

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14
Q

what is the pam sequence and its role?

A

very short sequence of nucleotides on dna
- signals cas9 to stop and check for complementary dna sequences to cut

no pam sequence=no cut=cannot be modified

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15
Q

benefits of the pam sequence

A
  • efficient: cas9 only looks for pams instead of searching through and unwrapping every dna sequence
  • protect bacteria dna: bacteria never have pam sequence in their own dna= dna cannot be cut up
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16
Q

2 ways crispr can be used to create genetically modified organisms

A

after cas9 cuts dna:
- gene knock in “editing”
- gene knock out “silencing”
= new nucleotides can be added to repair or silence target gene

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17
Q

what is gene knock in

editing

A

new dna sequence is inserted into the dna break
- allows faulty gene sequence to be replaced with correct sequence
= restore normal gene function

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18
Q

what is gene knock out

silencing

A

insertion/deletion of bases
- changes the way nucleotide sequences is read
= disables gene function or producing STOP signal
= silences faulty gene

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19
Q

technological uses of crispr?

A
  • correcting mutations responsible for disease
  • switching faulty genes off
  • adding new genes to an organism
  • studying the effects of specific genes
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20
Q

what is amplification

A

creates many copies of an original dna sample

repeat process many times

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21
Q

what is polymerase chain reaction?

A

dna amplification technique
- rapidly makes many copies of an original dna sample

2^x
x= number of cycles

22
Q

what is the purpose of amplification

A

increase quantity of identical copies of dna available
- large enough sample to be analysed

23
Q

what components are needed for pcr?

A
  • dna sample
  • dna polymerase (taq)
  • dna nucleotides
  • primers
  • mix buffer
  • pcr tube
  • thermal cycler

rna converted to dna- enzyme req for amp acts on dna,

24
Q

define target gene

A

gene of interest
- gene we want the bacteria to express

cut out of genome

25
Q

define plasmid

A

ring of dna

26
Q

what is a plasmid vector?

A

circular ring of dna
- used as a means of transporting the gene of interest into the bacteria

27
Q

what is a recombinant plasmid?

A

plasmid that has had the gene of interest inserted into it

28
Q

how is a recombinant plasmid created?

A
  • restriction enzyme cuts plasmid
  • same restriction enzyme removes target gene from dna
  • target gene and plasmid have complementary sticky ends
  • ligases join two pieces of dna

complementary sticky ends= easier to join

29
Q

explain the process of bacterial transformation with antibiotic resistance gene

A
  • gene of interest generated, inserted in appropriate bacterial plasmid vector chosen
  • bacteria are mixed with plasmids
  • heat/electroshock to encourage bacteria to take up recombinant plasmid
  • bacteria that take up recombinant plasmid= transformed
  • bacteria cultured on antibiotic medium
    = culture only transformed bacteria that will express gene of interest= can grow and form colonies
  • untransformed bacteria die

eg. gene for dystrophin is cut from human dna

  • plasmid vectors have antibiotic resistance gene
30
Q

what is the purpose of bacterial transformation? how can plasmids be used to make bacteria produce stuff

A

genetically modify bacteria to synthesis large amounts of protein
- transformed bacteria are grown/cultured and will transcibe and translate [gene of interest] producing [gene] which can then be harvested

31
Q

how could you use bacterial transformation to produce human insulin?

A
  • forgien dna and plasmid with amp resistance gene are cut in the middle of lacZ gene= produces b-galactosidase
  • bacteria cultured on a plate w amp and substance that changes colour when exposed to b-galactosidase
  • some cells do not take up plasmids= not amp resistant= dies/killed by amp
  • bacteria take up nonrecombinant with intact lacZ gene= survive antibiotic+changes colour
  • bacteria takes up recombinant plasmid= can’t produce b-galactosidace= survives+lacz no function= white colonies grow

lacZ: where restriction site is

32
Q

what is a reporter gene

A
  • distinguish between recombinant and non-recombinant plasmids
  • genes with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest
    eg. recognisable flourescent protein
33
Q

what are ways to use reporter genes to recognise bacterial transformation

A

reporter gene continuous: completely expressed
- bacteria that have been transformed with non recombinant plasmid to glow under UV
reporter gene noncontinuous: split by gene of interest
- bacteria taken up recombinant plasmid= cannot glow

34
Q

what are antibiotic resistance genes?

A
  • distinguishes between transformed and untransformed bacteria
  • only transformed bacteria contain the gene for antibiotic resistance= survives and multiplies
    eg. amp^r confers ampicillin resistance
35
Q

what are the steps and temps of pcr

A

denaturing: 95°
- strands seperate
annealing: 55°
- primers bind template
extending/elongation= 72°
- synthesis new strand

36
Q

describe the process of denaturing

A

-dna is heated to 95°
- breaks hydrogen bonds from heat
- 2 halves seperate= forms single stranded dna

37
Q

describe annealing in pcr

A
  • sample cooled to 55°
  • allows primers to bind to complementary sequences of single stranded dna
38
Q

what is a primer and its purpose (why are 2 needed)

A

short complementary single strand of nucleic acid
- acts as a starting point for dna polymerase to begin building dna strand
- provides taq polymerase with binding site to begin synthesis
(nucleotide sequences at 5’ end of the coding and template stands are different)

39
Q

what is elongation/extending in pcr

A
  • dna heated to 72°= taq polymerase work optimally
  • binds to primer
  • begins synthesising a new dna strand using complementary base pairs
40
Q

what is taq polymerase and why is it useful for pcr

A

type of dna polymerase
- does not denature at high degrees-> can maintain its shape and function at up to 95°
- optimal temp is high (72°)

41
Q

what is gel electrophoresis

A

technique used to compare dna samples by separating dna fragments according to their size

42
Q

what are the components of gel electrophoresis

A
  • dna samples placed in wells (negative electrode)
  • first well contains sample with dna fragments of known size (dna ladder)
  • electric current seperate the fragments
43
Q

why does dna from each sample appear in a series of bands on the gel

A
  • restriction enzymes cut the dna at a specifically recognised sequence
  • different individuals have different dna sequences= restriction enzyme will cut the dna in different places
    => results in fragments of varying size
44
Q

what direction does dna move through in gel electrophoresis

A

dna has overall negative charge= attracted towards + electrode, repelled from - electrode

45
Q

factors that affect the rate of movement of dna fragments through agarose gel

A
  • size/length of fragment (smaller dna fragments move quicker= travel further)
  • conc/viscosity of agarose
  • voltage/power used
    (NOT LENGTH OF TIME)
46
Q

what is the benefit of having degenerate codons

A

degree of redundancy
- chbaged to original dna sequence through mutations may not lead to insertion of different amino acid

47
Q

benefit of 2 types of genes onplasmid

A

another layer of selection
- let us know if bacteria is successfully transformed

48
Q

gmo def

A

genetically modified organism
- organism that has had its dna artificially altered in any way

49
Q

tgo def

A

transgenic organism
- organism that has had a gene from a different species inserted into its genome
(specific type of gmo)

50
Q

benefits of gmo?

A
  • increase crop yield= more nutritional value, store longer
  • decrease in pesticides, herbicides
  • drought, salt tolerance
  • improvement in health of humans, medicines
  • reduces cost and time associated with crop loss
51
Q

risks of gmo?

A
  • possible uncontrollable spread pf trans genes into other species
  • release of gmos into environment may be irreversible
  • animal welfare and ethical issues: poor health and reduced lifespan
  • emergence of pests, insects, microbial resistance to traditional control methods
  • monopoly and dependence of developing countries on company who are seeking to control seed supply of world

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