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BIOL 2320 - Genetics > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

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1
Q

Outline the steps required to clone DNA

A
  1. isolate donor and vector DNA
  2. cut donor and vector DNA with same restriction enzyme
  3. ligate donor and vector DNA together
  4. amplify recombinant DNA molecules in bacteria
  5. Select for bacteria that contains plasmid DNA
  6. Screen for bacteria containing a recombinant DNA molecule
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2
Q

Describe restriction enzymes

A

naturally occurring enzymes in bacteria that are used to cut both strands of the DNA sugar-phosphate backbone into two linear pieces

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3
Q

How do restriction enzymes work?

A

They scan along DNA until they reach the restriction sequence (GAATTC) and bind to DNA to break the covalent bond between GA on both strands

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4
Q

Describe vector

A

plasmid - accessory chromosomes that are not part of the bacterial chromosome

aka cloning vector

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5
Q

What is the purpose of the vector?

A

it is used to amplify the donor DNA in bacterial cell

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6
Q

What are 2 ways a restriction enzyme can cut the DNA strands?

A

Leaving single stranded overhangs by cutting at different locations on the strands = Make staggered cuts and leave ‘sticky’ ends

Leaving blunt ends by cutting at the same location on both strands

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7
Q

How do restriction enzymes work?

A

They scan along DNA until they reach the restriction sequence (ex. GAATTC for EcoRI) and bind to DNA to break the covalent bond between GA on both strands

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8
Q

Describe vector

A

AKA plasmid cloning vector = accessory chromosomes that are not part of the bacterial chromosome

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9
Q

What does ‘sticky’ end mean?

A

A result of how some restriction enzymes (ex. EcoRI) cut the DNA

they cut in a staggered way that leaves single stranded regions that are complimentary and when nearby, they can hydrogen bond to ‘stick’ back together

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10
Q

Describe genomic DNA

A

DNA obtained directly from chromosomes of the organism of interest

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11
Q

When is genomic DNA used in studies?

A

if the researcher wants to collect fragments of DNA that represent the entire genome of the organism

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12
Q

What does the vector contain that allows for the replication of DNA in bacteria?

A

origin of replication

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13
Q

What expression does the Amp^R gene have?

A

a selectable marker gene that gives bacteria ampicillin resistance

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14
Q

What is the function of the lacZ gene?

A

codes for an enzyme to break down Xgal (sugar) for the bacteria to digest

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15
Q

What is the multiple cloning site (MCS)?

A

a piece of DNA that contains a lot of restriction enzyme recognition sequences and is only found once in the plasmid

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16
Q

Where is the MCS located?

A

in the lacZ gene of the plasmid

17
Q

What is the multiple cloning site (MCS)?

A

a piece of DNA that contains a lot of restriction enzyme recognition sequences and is only found once in the plasmid (THE oNLY PLACE ENZYMES WILL CUT)

18
Q

What 4 things make up the vector/plasmid?

A

origin of replication (ori)

bla which produces Amp^R

lacZ which contains MCS

MCS

19
Q

T or F: the donor and vector DNA are cut with different enzymes?

A

False.

They are cut with the same restriction enzyme

20
Q

How are the donor and vector DNA cut with an enzyme?

A

by micropipetting

21
Q

How many times will the plasmid be cut? Where will it be cut?

A

Once

Only where the MCS is

22
Q

How many times will the donor DNA be cut? Where will it be cut?

A

thousands of times into tiny fragments of single stranded regions

cut wherever the restriction sequence occurs

23
Q

How do you ligate the donor and vector DNA together?

A

pipette some liquid from the donor DNA tube and some from the vector/plasmid DNA tube and combine into a third tube

Ideally they will find complementary single stranded regions and DNA ligase will form covalent bonds between the strands

24
Q

What enzyme will help in ligating the donor and vector DNA?

A

DNA ligase

25
Q

What is the most common method to get a recombinant DNA molecule into a bacterial host cell? why?

A

transformation

most bacteria are able to take up small fragments of DNA from surrounding environment

26
Q

In order for transformation to occur, what state do the cells need to be in?

A

they need to be competent

27
Q

How do you make cells competent?

A

adding salts and heating up will increase chances of the cells picking up the DNA from the environment

28
Q

What are the four different results of transformation?

A

Some bacteria will only take up the donor DNA

Some bacteria will only take up the vector/plasmid DNA

Some bacteria will take up no DNA

Some bacteria will take up the recombinant DNA (Both plasmid + donor DNA)

29
Q

What is the ideal result of transformation?

A

The bacterial cells taking up the recombinant DNA (donor + vector DNA)

30
Q

What is the ideal result of transformation?

A

The bacterial cells taking up the recombinant DNA (donor + vector DNA)

31
Q

Describe how DNA is amplified?

A

After transformation is successful,

replication will replicate a cells own genome + plasmid = large colony of bacteria

32
Q

After amplification, which bacterial cells are selected for? how?

A

bacteria with plasmid

Ampicillin is added to the medium to prevent the growth of the bacteria that do not have the plasmid

only bacteria with plasmid will have the Amp^R gene and be able to grow

this results in bacteria that took up only plasmid and bacteria that took up both

33
Q

How do you screen for bacteria containing a recombinant DNA molecule?

A

lacZ in the plasmid allows you to identify plasmids containing donor DNA (= recombinant DNA molecules)

34
Q

How does lacZ help identify recombinant DNA molecules?

A

lacZ gene produces enzyme that breaks down X-gal (sugar)

If lacZ gene is disrupted, there is donor DNA in the bacteria and it will not be able to break down X-gal (Recombinant)

35
Q

How can a researcher use lacZ to tell which bacteria are recombinant?

A

If lacZ is functioning, the bacterial colonies will be able to breakdown Xgal and the colonies will be blue

If lacZ is not functioning and was disrupted by donor DNA, it will not be able to breakdown Xgal and the colonies will be white

36
Q

What colour will the colonies be if the bacteria has recombinant DNA?

A

white

37
Q

Why do you have to screen for bacteria with recombinant DNA after selecting for AmpR?

A

Because 2 colonies will grow even after the ampicillin is added

One will be recombinant and one will be just carrying the plasmid

screening with lacZ will show which colonies ALSO have the donor DNA

BIOL 2320 - Genetics (12 decks)

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