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BIOL114 Biotechnology > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

Complete deck covering Lecture 1

1
Q

What are the stages in a typical DNA cloning experiment?

A
  1. Isolate and cleave DNA from two sources using the same restriction endonuclease
  2. Mix the two DNA segments together. Ligase joins them via their sticky ends
  3. Insert plasmid into bacterial cell via transformation. Cells grown on plate
  4. Clones screened for gene of interest
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2
Q

What is recombinant DNA

A

DNA that comes from multiple sources (e.g. two organisms) and combined in-vitro

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3
Q

True or False:

Genomic DNA is very long (>20kb fragments)

A

True

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4
Q

True or False:

Genomic DNA is used for genome mapping and sequencing

A

True

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5
Q

True or False:

Genomic DNA cannot be used for genome mapping and sequencing

A

False

Genomic DNA is used for genome mapping and sequencing

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6
Q

True or False:

Genomic DNA has no use for expressing protein in bacteria

A

True

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7
Q

True or False:

Genomic DNA is used for expressing protein in bacteria

A

False

Genomic DNA has no use for expressing protein in bacteria

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8
Q

True or False:

Eukaryotic genomic DNA contains introns

A

True

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9
Q

True or False:

Eukaryotic genomic DNA does not contain introns

A

False

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10
Q

True or False:

Bacterial genomic DNA contains introns

A

False

Bacterial genomic DNA does not contain introns

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11
Q

True or False:

Bacterial genomic DNA does not contain introns

A

True

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12
Q

When making recombinant DNA, what is the significance of bacterial genomic DNA not containing introns?

A

Bacteria cannot remove the introns from DNA so we have to use mRNA for the template (so that the introns have already been removed).

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13
Q

When using bacteria to make recombinant DNA, what is the significance of bacterial genomic DNA not containing introns?

A

Bacteria cannot remove the introns from DNA so we have to use mRNA for the template (so that the introns have already been removed).

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14
Q

What is a primer?

A

a short stretch of DNA complementary to the sequence

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15
Q

What does the ‘c’ stand for in cDNA?

A

Complementary

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16
Q

What is cDNA

A

Complementary DNA is artificially made, intron-lacking DNA

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17
Q

True or False:

All eukaryotic mRNAs have a poly-A tail in the three prime end

A

True

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18
Q

How is cDNA formed? (brief)

A

Single-stranded cDNA is synthesised from mRNA in vitro using the enzyme reverse transcriptase, a primer and the nucleotides dATP dGTP and dTTP

A second strand for stability is formed using a primer, nucleotides and the enzyme DNA polymerase as well as the first strand as a template

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19
Q

True or False:

We can use the poly-A tail in the three prime end of mRNA to generate a poly-T oligo (a complementary primer)

A

True

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20
Q

True or False:

We can use the poly-A tail in the three prime end of mRNA to generate an oligo dT (a complementary primer)

A

True

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21
Q

True or False:

We can use the poly-C tail in the three prime end of mRNA to generate a poly-T oligo (a complementary primer)

A

False

We can use the poly-A tail in the three prime end of mRNA to generate a poly-T oligo (a complementary primer)

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22
Q

True or False:

We can use the poly-C tail in the three prime end of mRNA to generate a poly-T oligo (a complementary primer)

A

False

We can use the poly-A tail in the three prime end of mRNA to generate a poly-T oligo (a complementary primer)

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23
Q

Fill in the gaps: (Making cDNA)
All eukaryotic mRNAs have a ____ tail in the three prime end. We can use this to generate a complementary primer called an _____.

The _____ will base pair with the _____ tail, and a _____________ enzyme that recognises this stretch of double stranded DNA and works in the direction of _____ prime to _____ prime can use the double stranded stretch of DNA to begin extending and synthesizing the full complementary strand using the mRNA as a template.

A

All eukaryotic mRNAs have a poly-A tail in the three prime end. We can use this to generate a complementary primer called an oligo.

The poly-T oligo will base pair with the poly-A tail, and a DNA polymerase enzyme that recognises this stretch of double stranded DNA and works in the direction of five prime to three prime can use the double stranded stretch of DNA to begin extending and synthesizing the full complementary strand using the mRNA as a template.

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24
Q

Fill in the gaps: (Making cDNA)
Once the DNA polymerase has extended and synthesised the full complementary strand using the mRNA as a template, an _______ _______ molecule DNA RNA is created.

It must become double stranded DNA to be stable so an _________ enzyme degrades the mRNA template and _______ _______ to add a poly-C stretch to the __ prime end of the remaining single stranded DNA molecule.

A

Once the DNA polymerase has extended and synthesised the full complementary strand using the mRNA as a template, an unstable hybrid molecule DNA RNA is created.

It must become double stranded DNA to be stable so an RNAse H enzyme degrades the mRNA template and terminal transferase to add a poly-C stretch to the 5 prime end of the remaining single stranded DNA molecule.

25
Q

Fill in the gaps: (Making cDNA)
A complimentary primer _________ binds to the poly-C stretch to create short double stranded DNA.

A DNA polymerase travelling in the __ prime to __ prime direction can use this as an anchor to begin extending and synthesizing the complementary strand to our template and generate the full double strand of cDNA, making it ______.

A

A complimentary primer poly-G oligo binds to the poly-C stretch to create short double stranded DNA.

A DNA polymerase travelling in the 5 prime to 3 prime direction can use this as an anchor to begin extending and synthesizing the complementary strand to our template and generate the full double strand of cDNA, making it stable.

26
Q

Where can you find bacterial extrachromosomal DNA?

A

In it’s plasmids

27
Q

List different vectors which can be used for recombinant DNA

A

Plasmids (most common)
Viruses
Phages
Yeast

28
Q

Where did restriction endonucleases originate from?

A

Restriction endonucleases are a bacterial defence against viral infections, we get them by isolating them from bacteria

29
Q

What is the correct name of restriction enzymes?

A

Restriction endonucleases

30
Q

How do bacteria protect their own DNA from their restriction endonucleases?

A

Bacteria have additional methyl groups to the A’s and C’s within the sequence recognised by the restriction endonucleases, since they cannot cut methylated DNA

31
Q

True or False:

There are lots of different kinds of restriction endonucleases which chop DNA at defined sites

A

True

32
Q

True or False:

There is only one type of restriction endonuclease that we use to chop DNA at defined sites

A

False

There are lots of different kinds of restriction endonucleases which chop DNA at defined sites

33
Q

How are endonucleases named?

A

They are named after the bacterium species they were isolated from

34
Q

Which endonuclease was first isolated from E. coli?

A

EcoRI

35
Q

Which endonuclease was first isolated from Arthrobacter luteus?

A

AluI

36
Q

Fill in the gaps:
Restriction endonucleases recognise sequences which may be __ , __, or __ nucleotides long.

The recognition sequences have two-fold _______ _________, gaining the name of ________ sites.

A

Restriction endonucleases recognise sequences which may be 4 , 6, or 8 nucleotides long.

The recognition sequences have two-fold rotational symmetry, gaining the name of palindromic sites.

37
Q

How do you calculate the probability / how often a restriction endonuclease will cut a piece of DNA?

Why is this useful?

A

1/4 ^ (number of bases in recognition sequence)
Gives how often on average a cut takes place.

Eg; 4 base recognition sequence:
1/4 ^4 = 1/256 so cuts on average every 256 base pairs

Eg; 6 base recognition sequence:
1/4 ^6 = 1/4096 so cuts on average every 4096bp

Useful because can choose which base sequence recognition we use depending on how often we want it cut (how large or small we want the DNA pieces)

38
Q

What are blunt ends?

A

DNA is cut in the middle of a sequence.

Not as commonly used as sticky ends but for some DNA this is the only way to replicate it.

39
Q

How are sticky ends formed?

A

Restriction endonuclease cuts sugar-phosphate bond between the same two nucleotides (eg; A and G) in each strand - ‘same place’. Since section is palindromic, leaves the strands with sticky ends, a sort of overlap.

40
Q

True or False:
The new piece of DNA is inserted between the sticky ends of the plasmid DNA and DNA Ligase recombines the cuts made to join them all together

A

True

41
Q

True or False:

Restriction endonucleases cut the sugar-phosphate backbone

A

True

42
Q

True or False:

Restriction endonucleases cut the hydrogen bonds between the DNA strands at specific points in the sequence

A

False

Restriction endonucleases cut the sugar-phosphate backbone

43
Q

How does DNA ligase reform the backbone of the DNA to join everything together?

A

DNA ligase ligates the phosphodiester backbone by joining the 5 prime phosphate to the 3 prime hydroxyl group in the double stranded DNA

44
Q

Fill in the gaps:
Vectors for DNA fragments are usually relatively _____ DNA molecules, usually with ____________ for the chosen restriction enzyme.

The vectors should be capable of ___________________ and contain a _________ gene (such as ______ _______)

A

Vectors for DNA fragments are usually relatively small DNA molecules, usually with a single site for the chosen restriction enzyme.

The vectors should be capable of replicating in a host organism and contain a selectable gene (such as antibiotic resistance)

45
Q

Fill in the gaps:
The plasmids we use have a ___________ site, with sequences for ______ ________ restriction enzymes.

This means we can choose an appropriate enzyme depending on the piece of DNA, ______________ and the restriction fragments we can generate.

A

The plasmids we use have a multi-cloning site, with sequences for many different restriction enzymes.

This means we can choose an appropriate enzyme depending on the piece of DNA, our gene of interest and the restriction fragments we can generate.

46
Q

List different hosts which can be used for recombinant DNA

A

Bacteria (modified to eliminate pathogenic qualities)
Yeast
Animal cells in culture

47
Q

What properties must the host have to be viable for use in recombinant DNA processes?

A

Must be able to be grown easily in large quantities

Must be non-pathogenic
so if bacteria is used, for example, must be modified to eliminate pathogenic qualities

48
Q

Fill in the gaps:
The process of delivering the plasmid to the host cell is called ___________.

Bacterial cells, called __________ cells, are treated to make their membranes _____________ in certain conditions, such as when _____ is applied. Warming these cells loosens their membranes to allow plasmids in.

____________ is another process that creates similar pores in the membranes to allow the plasmids to permeate into the cell.

A

The process of delivering the plasmid to the host cell is called transformation.

Bacterial cells, called competent cells, are treated to make their membranes more permeable in certain conditions, such as when heat is applied. Warming these cells loosens the membranes to allow plasmids in.

Electroporation is another process that creates similar pores in the membranes to allow the plasmids to permeate into the cell.

49
Q

What is a relitigated plasmid?

A

After being cut by the restriction enzyme, the DNA re-circularized, so the sticky ends relitigated (re-joined) before taking up the insert of the gene of interest

50
Q

A bacterial cell must have a plasmid for a chance to take up the gene of interest.

How can we tell which cells contain a plasmid (regardless of if the plasmid contains the insert of the gene of interest)?

A

A selectable gene (eg; antibiotic resistance) is inserted with the gene containing recombinant DNA. This is used to identify the plasmids containing the gene of interest.

E.g. antibiotic resistance, use a specific antibiotic to kill all vectors not containing the antibiotic resistance gene which is only carried in the plasmid. Only vectors with resistance have a chance of carrying the recombinant DNA.

51
Q

Give two examples of antibiotic resistance

A

Ampicillin (Amp^R)

Kanamycin

52
Q

Fill in the gaps:
A plasmid cut with restriction enzymes is referred to as a ______ plasmid, because when its cut it is not _______ anymore.
Treating this plasmid with _______ __________ removes __________ ______ from the end of the plasmid.
The only way to regenerate the sugar phosphate backbone is if insert carries _________ ______ to pair with the ______ ______ from the vector.
This treatment minimizes probability that the plasmid recircularizes without the insert.
We cannot fully prevent the recircularization of the plasmid with no insert.

A

A plasmid cut with restriction enzymes is referred to as linear plasmid, because when its cut it is not circular anymore.
Treating this plasmid with alkaline phosphatase removes phosphate groups from the end of the plasmid.
The only way to regenerate the sugar phosphate backbone is if insert carries phosphate groups to pair with the hydroxyl groups from the vector.
This treatment minimizes probability that the plasmid recircularizes without the insert.
We cannot fully prevent the recircularization of the plasmid with no insert.

53
Q

Fill in the gaps: (Blue-white screening)
Vector contains a gene which codes for the enzyme _______________ (in addition to carrying an antibiotic).
This enzyme converts the substrate_____ into a blue-coloured product, meaning our cells/vectors can turn blue.
If this gene is _______ by the insertion of a DNA fragment, when cells are plated in media containing X-gal:
___________________________
___________________________
___________________________

A

Vector contains a gene which codes for the enzyme ß-galactosidase (lacZ) (in addition to carrying an antibiotic).
This enzyme converts the substrate X-gal into a blue-coloured product, meaning our cells/vectors can turn blue.
If this gene is disrupted by the insertion of a DNA fragment, when cells are plated in media containing X-gal:
no enzyme will be produced in cells with gene of interest
no blue product is produced so white colonies contain gene of interest
identification of recombinant clone

54
Q

In the context of recombinant DNA, what is a library?

A

Large number of different genomic fragments or cDNAs inserted into vector
Mixture of recombinant DNAs, each with a different insert
Each colony is derived from a single recombinant DNA molecule
Library becomes a collection of clones

55
Q

What is the name of the process we use to identify the clone containing our gene of interest in a library?

A

Colony hybridisation

56
Q

Why does blue-white screening not identify which colonies have the gene of interest in a library?

A

Each white colony will carry a different gene

57
Q

Fill in the gaps: (Colony hybridisation)
A _____ (small piece of DNA, part of our sequence of interest) is generated and _______, usually with __________ ___________. During the generation of the _____, one nucleotide is _________ _______ with ___ is incorporated.
For libraries, a _____ is used to transfer and _________ some cells, containing bacteria from all the colonies we have on the plate. The _____ is treated with _____ solutions to generate single stranded DNA (_______).

A 
A probe (small piece of DNA, part of our sequence of interest) is generated and labelled, usually with radioactive phosphorus. During the generation of the probe, one nucleotide radioactively labelled with P32 is incorporated.
For libraries, a filter is used to transfer and immobilize some cells, containing bacteria from all the colonies we have on the plate. The filter is treated with salt solutions to generate single stranded DNA (denatured).
58
Q

Fill in the gaps: (Colony hybridisation)
The solution containing the single stranded ____________ _____ for the gene of interest is added to the filter.
The _____ _______ to the complementary part of the gene of interest in hybridisation.
The _____ is ___________, so positives can be identified by ______________ (x ray film on top of membrane).
Comparing the signal detected from this with the master plate allows identification of colonies of interest in our bacterial plate.

A

The solution containing the single stranded radioactive probe for gene of interest is added to the filter.
The probe anneals to the complementary part of the gene of interest in hybridisation.
The probe is radioactive, so positives can be identified by autoradiography (x ray film on top of membrane).
Comparing the signal detected from this with the master plate allows identification of colonies of interest in our bacterial plate.

59
Q

Define Biotechnology

A

“The manipulation (as through genetic engineering) of living organisms or their components to produce useful, usually commercial, products (as pest resistant crops, new bacterial strains, or novel pharmaceuticals)”

“Any of various applications of biological science used in such manipulation”

BIOL114 Biotechnology (10 decks)

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