The technology underpinning molecular biology: gene cloning Flashcards

1
Q

For what downstream purpose is cloning genes necessary?

A

to study, modify or transfer a particular gene (or fragment of DNA

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2
Q

What is gene cloning?

A

identification and replication of DNA at a high copy number, in a host or in vitro

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3
Q

To be replicated in a host, DNA must

A

be a replicon

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4
Q

replicon

A

contain a replication origin

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5
Q

Most DNA fragments are not replicons & thus

A

would be diluted during host cell division

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6
Q

How is the replicon issue solved?

A
  • plasmids and bacteriophages are replicons
  • used as “vectors”
  • enable replication of a single DNA fragment in a host cell (or clone of cells)
  • generate a library of many cells/clones, each one containing a different fragment
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7
Q

Describe the function of a gene library

A

screened to identify the clone carrying the DNA of interest

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8
Q

Describe PCR

A

used to replicate DNA at a high copy number in vitro

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9
Q

Describe gene cloning

A
  • construct different recombinant DNA molecules using DNA fragments
  • introduce into a bacterial host
  • plate out onto an agar medium, to grow colonies or clones of identical host cells
  • grow at 37 degrees
  • each colony contains multiple copies of just one recombinant DNA molecule
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10
Q

Give an example of a bacterial host used in gene cloning

A

E. coli

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11
Q

Describe phage resistant bacteria

A
  • host DNA is modified and therefore protected, by cleavage phage DNA
  • no plaques form
  • restricting bacteria - endonuclease
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12
Q

Describe susceptible bacteria

A
  • phage offspring released then cell is injected with phage DNA by bacteriophage
  • cycles of cell lysis and localised reinfection form plaques
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13
Q

How is DNA cut with restriction enzymes?

A
  • palindromic recognition site (4,6 or 8bps long)
  • binding
  • cleavage generates cohesive or blunt termini
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14
Q

Describe the relationship between recognise site length and cut frequency

A
  • 4 bp cutters cut more frequently
  • 8 bp cutters cut less frequently
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15
Q

Give the name of a restriction endonuclease

A

EcoRI

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16
Q

cohesive termini

A

sticky ends

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17
Q

How is the cut DNA joined?

A
  • hydrogen bonds form between cohesive termini
  • phosphodiester bond formation catalysed by DNA ligase in ATP-dependent reaction
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18
Q

Describe the DNA ligase molecule used in cloning

A
  • T4 DNA ligase
  • typically from bacteriophage T4
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19
Q

What is the function of gel electrophoresis?

A

separating DNA fragments based on size

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20
Q

How does gel electrophoresis work?

A
  • agarose gel immersed in buffer, placed in an electric field
  • DNA samples loaded into wells at one end of the gel
  • fragments migrate towards the anode
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21
Q

Describe intercalating agent in gel electrophoresis

A
  • e.g. EtBr
  • inserts itself between DNA bases and fluoresces
  • can be viewed under UV transilluminator
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22
Q

Describe the allele ladder

A
  • DNA fragments of known sizes, purchased commercially
  • used to size the fragments of interest
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23
Q

What are the two types of libraries?

A
  • genomic library
  • cDNA library
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24
Q

Describe a genomic library

A
  • used if interested in the whole transcription unit or intergenic regions
  • contains all the DNA sequences in a cell
  • can be >90% non-coding
  • sample DNA must be fragmented before cloning
  • fragment / insert sizes typically large (e.g.~15-20 kb if using phage vector)
  • high number of repetitive sequences
  • size of library required depends on genome size
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25
Describe DNA fragmentation before cloning in a genomic library
- neither practical nor helpful to clone an entire chromosome - partial restriction enzyme digestion or random shearing
26
What does the quality of a genomic library depend on?
- quality of fragmentation - need maximum number of large, overlapping fragments
27
Describe a cDNA library
- required if only interested in mRNA sequence - made from mRNA of a particular tissue-type or time-point - inserts are DNA, but are complementary to the mRNA - sample fragmentation not required - fragment / insert sizes typically small (~0.5-5 kb) - may not contain all gene sequences - contains few repetitive sequences - size of library needed depends on relative abundance of mRNA of interest
28
What affects the quality of a cDNA library?
- quality of the mRNA isolated - gene expression profile of sampled tissue
29
When would a plasmid vector be chosen?
- 5,000bp insert size - 1,800,000 clones to represent human genome
30
When would a phage vector be chosen?
- 20,000bp insert size - 450,000 clones to represent human genome
31
When would a cosmid vector be chosen?
- 40,000bp insert size - 225,000 clones to represent human genome
32
When would a BAC vector be chosen?
- 150,000bp insert size - 60,000 clones to represent human genome
33
When would a YAC vector be chosen?
- 300,000bp insert size - 30,000 clones to represent human genome
34
cosmid
- plasmid with cos sites - allows packaging into phage particles - more efficient delivery
35
BAC
-bacterial artificial chromosome - based on F plasmid that can carry v. large inserts at low copy no.
36
YAC
yeast artificial chromosome
37
Describe plasmid vectors
carry inserts up to 5-10kb
38
Describe puC18/19
- encodes beta-galactosidase - ori, MCS, lacZ, AmpR - creates blue product from colourless X-gal - allows for blue-white selection on an X-gal plate
39
AmpR
selectable marker
40
Describe phage vectors
- carry inserts up to 20kb
41
Describe phage lambda
- 48,502nta - 20kb dispensable, stuffer fragment required for lysogen, but not important for vector use - 12bp cohesive ends at each arm enable packaging and circularisation in host - creates plaques
42
Describe YAC vectors
- shuttle vectors that can be propagated in bacteria to prepare vector for cloning, or in yeast to carry large inserts - carry inserts up to 300kb
43
Describe circular YAC vector
- e.g. pYAC3 - cloning site (EcoRI) - inserted here
44
Describe the preparation of a genomic library in a phage
- genomic DNA partially digested with restriction enzyme - stuffer fragment removed from bacteriophage lambda - genomic fragments ligated into vector arms - concaterner of many recombinant phages cleaved - packaged into infective phage particles - infected into E. coli - plaques develop
45
Describe the results of a phage genomic library
- each plaque correspond to a single clone - each clone carries a single fragment of genomic DNA from the genome of interest
46
Describe the purification of mRNA for cDNA synthesis
- total cellular RNA isolated from cells and passed through elution column - poly-A tails of mRNA pair with oligo(dT) chains - mRNA retained in column - other RNAs pass through
47
Describe total cellular RNA
- prepared by treatment of cell lysate with organic solvents to remove protein/DNA - or using a commercial kit
48
Elution column
contains oligo(dT) chains linked to cellulose
49
oligo(dT)
short, single-stranded DNA sequence containing only thymine nucleotides
50
cellulose
inert matrix
51
mRNAs
have poly-A tails
52
When you purify RNA from cells, it is
a complex mixture containing rRNA, tRNA and mRNA et al.
53
What is the function of the buffer in the elution column?
breaks the hydrogen bonds between poly-A tails and oligo(dT) chains
54
cDNA synthesis requires
- purified mRNA - dscDNA
55
Describe the first strand of cDNA synthesis
- mRNA template - oligo-dT primers added and anneals - 4x dNTPs and reverse transcriptase added - incubation - creates sscDNA
56
dNTPs
nucleoside triphosphates
57
Describe the second strand cDNA synthesis
- RNaseH generates nicks in mRNA - second strand synthesis begins with DNA polymerase I - second strand conclusion with T4 DNA ligase
58
DNA polymerase I
exonuclease
59
Why are library plate replicas required?
- easy to screen - viable master plate can be preserved for future propagation
60
Describe the replication of library plates
- nitrocellulose disc overlayed onto master plate containing clone library inside E. coli cells - binds DNA efficiently to make a replica - disc removed and cells lysed with NaOH (denatures DNA) - baked and treated with UV light - DNA strands bind to disc - ready for screening
61
How are probes made?
- labelling of a DNA fragment - purified DNA corresponding to, or similar to fragment of interest forms a template - denatured by heating to 95 degrees, then following - mixture of random hexanuclotide primers anneal - add labelled dNTPs and Klenow fragment of DNA polymerase I - denatured using heat to form a ss - incorporation of labelled nucleotide means that it is a probe
62
Name some ways of labelling dNTPs
- radioactive - fluorescent - enzyme-linked detection tag
63
Describe radioactive screening by DNA hybridisation
- DNA on replica nitrocellulose filter - radioactive probe added - replica hybridised, washed and dried - labelled probe forms duplex with cDNA - autoradiography
64
Describe autoradiography
- exposure to X-ray film - detects radioactivity - spot on film locates colony - related to the master plate to identify the clone of interest - clone propagated
65
Describe PCR
- DNA is heated to 90-100 degrees to separate the two strands - DNA quickly cooled to 30-65 - short, ss primers anneal to complementary sequences - heated to 60-70 degrees - DNA polymerase synthesises new DNA strands - two new, dsDNA molecules created - cycle repeats, doubling DNA each time
66
What are the ingredients necessary for PCR?
- template DNA - 2x ssDNA oligonucleotide primers - 4x dNTPs - taq polymerase - thermal cycler
67
From where is taq polymerase derived?
Thermus aquaticus
68
Describe PCR
- much faster than library screening - requires prior knowledge of target for primer design - in vitro method - products can be cloned in vectors for further propagation and manipulation
69
How long does library screening take?
several weeks