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BioTech Martin Edwards > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

How can we denature DNA?

A

Heat
Extreme pH
Hydrogen bond breaking agents e.g. concentrated Urea

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2
Q

Under similar conditions the temp at which a (long) DNA double strand will denature depends on

A

its base composition

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3
Q

DNA with a higher G+C content will denature at a …..

A

higher temperature

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4
Q

Why would DNA with higher G+C content denature at a higher temperature than DNA with a lower G+C content?

A

G+C held together more strongly (3 hydrogen bonds) than A+T is (2 hydrogen bonds)

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5
Q

What conditions favour double helix formation?

A

Altered temperature
High salt conc (ionic strength)
Removal of denaturants

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6
Q

Hybridisation can only occur between DNA strands which are

A

complementary to each other

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7
Q

What happens in membrane-hybridisation assay?

A

Melt ds DNA to ss DNA, DNA binds to filter, incubate filter with labelled DNA, wash away labelled DNA that did not hybridise to DNA bound to filter.

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8
Q

What can you use southern blotting for?

A

Determine how many genes correspond to cDNA probe are present in organisms genome

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9
Q

What happens in southern blotting?

A

1) DNA cleaved with restriction enzymes
2) Gel electrophoresis
3) Place filter paper over nitrocellulose and gel in alkaline solution
4) Alkaline solution denatures DNA, capillary action transfers DNA from gel to nitrocellulose
5) Hybridise with labelled DNA probe and examine e.g. using autoradiogram

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10
Q

What are northern blots used for?

A

Standard method of analysing mRNA, gives size and amount of RNA.

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11
Q

What happens in northern blotting?

A

1) unlabelled RNA, undergoes agarose gel electrophoresis where nucleic acids are separated according to size
2) separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper
3) nitrocellulose paper has labelled probe hybridised to its complementary bands
4) visualised by autoradiography

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12
Q

What is PCR used for?

A

Amplifying DNA sequences

  • cloning
  • probes
  • forensics
  • PCR can be used to amplify rare DNA from a mix (when end sequences known)
  • amplification of mutant alleles allows detection of human disease
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13
Q

Requirements of PCR

A

Taq DNA polymerase
dNTPS
Oligonucleotide primers
Mg2+ ions

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14
Q

What does Taq DNA polymerase do?

A

Copies original and generates new strands

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15
Q

why are dNTPS needed in PCR?

A

they are nucleotide bases that form the new DNA strands

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16
Q

Why are oligonucleotide primers needed in PCR?

A

forms initiation site for Taq polymerase

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17
Q

Why are Mg2+ ions needed in PCR?

A

stabilise the primer

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18
Q

What are the requirements for the design of a PCR primer?

A
  • primers should be 20 bases long
  • The G/C content should be 45-55%
  • The annealing temps should be within 1 degree of each other
  • The 3’- most base should be a G or C (as 3 hydrogen bonds will clamp tight)
  • Primers must not base pair with self or other primers to form hairpins
  • Primers must avoid repetitive DNA regions (you’d get multiple binding of primers)
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19
Q

How can you optimise the annealing temp of PCR?

A

-Primers have a concentrated annealing temp, this temp must be confirmed practically. You can test temps of 2 above and below using gradient cycler.

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20
Q

How can you optimise the Mg2+ conc of PCR?

A

The fidelity of PCR depends on Mg2+

  • Vary Mg2+ in steps of 0.5mM
  • Sometimes have to compromise between yield and specificty
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21
Q

How can you optimise the PCR reaction?

A

CONSIDER

  • annealing temp of primers
  • Conc of Mg2+ in reaction
  • The extension time
  • The amount of template
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22
Q

Why is it difficult to find an optimum annealing temp for primers?

A

At low temperatures you get non-specific binding and whilst this would improve with higher temps, high temps can reduce yield

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23
Q

How do primers form hairpins?

A

If a primer is self-complementary it can fold into a hairpin, the 3’ end of the primer is therefore base-paired so it can’t anneal to target DNA

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24
Q

How do primers form dimers?

A

A primer can form a dimer with itself or other primers. Primer dimers are used as a substrate for Taq polymerase (amplifies this and not target DNA)

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25
Q

How can you use PCR to modify a target sequence to aid cloning?

A

As long as 3’ end region is exact to binding can do what you want to 5’ end
Can use this to engineer extra information into DNA

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26
Q

The first step of TCR is to heat the mixture to 95 degrees, why?

A

ds DNA denatured to ss DNA as hydrogen bonds that hold the two polynucleotide strands of DNA together are broken.

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27
Q

What is the third temp in PCR that allows DNA synthesis to begin and is just below the optimum for Taq polymerase?

A

74 degrees

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28
Q

What is the second temperature that allows primers to attach to their annealing position?

A

55-60 degrees

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29
Q

What could happen if the primer is too short?

A

It may hybridise to non-target sites and give undesired amplification products

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30
Q

Why can you not just use a really long primer?

A

Length influences rate of hybridisation to DNA and therefore PCR efficiency

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31
Q

What will happen if the temperature is too high?

A

No hybridisation will occur and primers and template will remain dissociated

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32
Q

What will happen if the temperature is too low?

A

Amplification is less likely to occur at target site so get hybrids with incorrect base pairing

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33
Q

How can the perfect annealing temp be calculated?

A

Perfect temp can be estimated by determining the Tm of primer-template hybrid (a temp 1-2 degrees lower will be right)

34
Q

Why can’t PCR products just be placed in a normal vector for cloning?

A

The ends are not blunt but instead have a single nucleotide hangover at most of 3’ termini.

35
Q

What special kind of vector can you use for PCR products?

A

A special cloning vector which carries thymidine overhangs that can be ligated to PCR product.

36
Q

What are the two common vectors?

A

E.coli plasmid vectors and bacteriophage vectors

37
Q

Bacterial plasmids are … occurring ….. ds DNA molecules

A

naturally … circular

38
Q

Bacterial plasmids are extra….

A

chromosomal

i.e. separate from the main genomic DNA of the organism

39
Q

What size are bacterial plasmids?

A

200kb

40
Q

What is the function of bacterial plasmids in nature?

A

enhance adaptation of bacteria to environment by transferring genes horizontally

41
Q

What are bacterial plasmids responsible for?

A

Outbreaks of antibiotic resistance pathogenic bacteria in hospitals
Due to transfer of plasmids containing antibiotic resistance genes between bacterial spp

42
Q

What two vital features do plasmids contain?

A

A replication of origin

A gene which confers a selective advantage and selects for the plasmid

43
Q

What does the replication of origin enable the plasmid to do?

A

Be replicated independently of the bacterial genome

44
Q

Plasmids are passed on to

A

daughter cells when fission of bacterial cells occur

45
Q

Only one plasmid of a given replication group can be

A

stably maintained in a bacterial cell

46
Q

What determines the replication group of the plasmid?

A

The origin of replication

47
Q

What determines the plasmid copy of a cell?

A

The origin of replication

48
Q

Can plasmids from different replication groups be stably maintained in a bacterial cell simultaneously?

A

yes

49
Q

Why are plasmids spontaneously lost?

A

Replication of a plasmid produces a metabolic load the host organism so if there is no selective advantage they will be spontaneously lost

50
Q

Purification relies on physical properties of plasmid DNA such as what?

A
  • different sedimentation in density gradient
  • different behaviour on denaturation
  • ion exchange behaviour
51
Q

Plasmids can be purified quite easily from bacteria after cell lysis but yields are

A

LOW

52
Q

What is the result of bacterial cell after lysis?

A

circular ds DNA molecule- usually with added supercoil

53
Q

How are plasmids reintroduced into bacteria by transformation?

A
  • Bacterial cells made leaky by treatment with membrane permeabilising agents
  • These are known as competent
  • Competent cells mixed with DNA and subjected to mild heat shock
  • DNA uptake can be increased by electroporation
54
Q

DNA is taken up at …. efficiency

A

very low

55
Q

Efficiency transformation falls rapidly as

A

size of circular DNA increases

56
Q

What is the general procedure for cloning with plasmid vectors?

A

1) Enzymatically insert DNA into plasmid vector
2) Mix E.coli cell with plasmids in presence of CaCl2
3) Culture on nutrient agar plates containing ampicillin
4) Transformed E.coli cells survive
5) Cells that do not take up plasmid die on ampicillin plate
6) Independent plasmid replication

57
Q

What is the process of Blue/white selection?

A

1) The host E.coli cell carries the w-peptide whilst the plasmid carries the a-peptide
2) When the two peptides are expressed together they form active B-galactosidase
3) Cells with original plasmid produce a blue colony when treated with X-Gal.
3) )In plasmids with an insert, the gene that produces a-peptide is disrupted so no function B-galactosidase can be formed so colinies are white

58
Q

Cells with the original plasmid will be what colour when treated with X-Gal?

A

Blue

59
Q

In plasmids with an insert a-peptide is mutated and the colonies are what colour?

A

White

60
Q

What would a designer plasmid have vector have?

A
  • Ori : origin of replication
  • Ampr: ampicllin resistance genes
  • MCS: multi purpose cloning site inside lacZ gene
  • lacZ: cloning sequence for B-galactosidase a-peptide (allows for blue white selection)
61
Q

What size is desirable for a cloning vector?

A

10kb

62
Q

What does the copy number refer too?

A

The number of molecules of an individual plasmid that are normally found in a bacterial cell

63
Q

Stringent plasmids have a ….. copy number

A

low

64
Q

Relaxed plasmids have a …. copy number

A

high

65
Q

What are the types of plasmid according to classification?

A
  • F plasmids
  • R plasmids
  • Ti plasmids
66
Q

What is the best characterised eukaryotic plasmid?

A

2um circle occurs in Saccharomyces cerevisiae

67
Q

What are Cosmid vectors?

A

A plasmid containing a cos site, so DNA can be packaged into phage particles in vitro

68
Q

Why can Cosmid vectors carry larger DNA inserts than bacteriophage vectors?

A

There are no bacteriophage genes

69
Q

Cosmid vectors infect like …. viruses and ….. like plasmids

A

viruses and replicate

70
Q

What are YACs?

A

Yeast artificial chromosomes

71
Q

What are YACS used for?

A

-Very large DNA inserts

72
Q

What essential elements of a yeast chromosome do plasmids include?

A

-DNA replication origin
-Centromere
-Telomere
(Thus insert will replicate in yeasr cells as part of an artificial chromosome)

73
Q

What are BAC vectors?

A

bacterial artificial chromosomes

74
Q

BACs can hold inserts up to

A

300kbp

75
Q

How many copies of BAC vectors do you get per cell?

A

1 or 2 copies.

76
Q

The 2um is an excellent cloning vector it is …. in size and exists in yeast at cell at copy number …. replication makes use of the …. and the proteins coded by ….. genes.

A

6kb
70 and 200
Origin of replication
REP1 and REP2

77
Q

Vectors derived from the 2um plasmid are called

A

Yeps

78
Q

In adiition to YEps what are the other types of clonal vectors for use with S.Cerevisiae

A

YIps and YRps

79
Q

What factors come into play when deciding which type of yeast vector s most suitable for cloning experiment?

A 
Transformation frequency (necessary if large number of recombinants needed)
Copy Number
80
Q

YIps have a very low copy number BUT

A

but produce very stable recombinants

81
Q

YRps recombinants are very unstable but

A

have a high transformation frequency

BioTech Martin Edwards (3 decks)

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