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Hilary Term 1 > The technology underpinning molecular biology: gene cloning > Flashcards

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
Q

Describe DNA fragmentation before cloning in a genomic library

A
  • neither practical nor helpful to clone an entire chromosome
  • partial restriction enzyme digestion or random shearing
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26
Q

What does the quality of a genomic library depend on?

A
  • quality of fragmentation
  • need maximum number of large, overlapping fragments
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27
Q

Describe a cDNA library

A
  • 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
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28
Q

What affects the quality of a cDNA library?

A
  • quality of the mRNA isolated
  • gene expression profile of sampled tissue
29
Q

When would a plasmid vector be chosen?

A
  • 5,000bp insert size
  • 1,800,000 clones to represent human genome
30
Q

When would a phage vector be chosen?

A
  • 20,000bp insert size
  • 450,000 clones to represent human genome
31
Q

When would a cosmid vector be chosen?

A
  • 40,000bp insert size
  • 225,000 clones to represent human genome
32
Q

When would a BAC vector be chosen?

A
  • 150,000bp insert size
  • 60,000 clones to represent human genome
33
Q

When would a YAC vector be chosen?

A
  • 300,000bp insert size
  • 30,000 clones to represent human genome
34
Q

cosmid

A
  • plasmid with cos sites
  • allows packaging into phage particles
  • more efficient delivery
35
Q

BAC

A

-bacterial artificial chromosome
- based on F plasmid that can carry v. large inserts at low copy no.

36
Q

YAC

A

yeast artificial chromosome

37
Q

Describe plasmid vectors

A

carry inserts up to 5-10kb

38
Q

Describe puC18/19

A
  • 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
Q

AmpR

A

selectable marker

40
Q

Describe phage vectors

A
  • carry inserts up to 20kb
41
Q

Describe phage lambda

A
  • 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
Q

Describe YAC vectors

A
  • 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
Q

Describe circular YAC vector

A
  • e.g. pYAC3
  • cloning site (EcoRI) - inserted here
44
Q

Describe the preparation of a genomic library in a phage

A
  • 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
Q

Describe the results of a phage genomic library

A
  • each plaque correspond to a single clone
  • each clone carries a single fragment of genomic DNA from the genome of interest
46
Q

Describe the purification of mRNA for cDNA synthesis

A
  • 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
Q

Describe total cellular RNA

A
  • prepared by treatment of cell lysate with organic solvents to remove protein/DNA
  • or using a commercial kit
48
Q

Elution column

A

contains oligo(dT) chains linked to cellulose

49
Q

oligo(dT)

A

short, single-stranded DNA sequence containing only thymine nucleotides

50
Q

cellulose

A

inert matrix

51
Q

mRNAs

A

have poly-A tails

52
Q

When you purify RNA from cells, it is

A

a complex mixture containing rRNA, tRNA and mRNA et al.

53
Q

What is the function of the buffer in the elution column?

A

breaks the hydrogen bonds between poly-A tails and oligo(dT) chains

54
Q

cDNA synthesis requires

A
  • purified mRNA
  • dscDNA
55
Q

Describe the first strand of cDNA synthesis

A
  • mRNA template
  • oligo-dT primers added and anneals
  • 4x dNTPs and reverse transcriptase added
  • incubation
  • creates sscDNA
56
Q

dNTPs

A

nucleoside triphosphates

57
Q

Describe the second strand cDNA synthesis

A
  • RNaseH generates nicks in mRNA
  • second strand synthesis begins with DNA polymerase I
  • second strand conclusion with T4 DNA ligase
58
Q

DNA polymerase I

A

exonuclease

59
Q

Why are library plate replicas required?

A
  • easy to screen
  • viable master plate can be preserved for future propagation
60
Q

Describe the replication of library plates

A
  • 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
Q

How are probes made?

A
  • 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
Q

Name some ways of labelling dNTPs

A
  • radioactive
  • fluorescent
  • enzyme-linked detection tag
63
Q

Describe radioactive screening by DNA hybridisation

A
  • DNA on replica nitrocellulose filter
  • radioactive probe added
  • replica hybridised, washed and dried
  • labelled probe forms duplex with cDNA
  • autoradiography
64
Q

Describe autoradiography

A
  • 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
Q

Describe PCR

A
  • 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
Q

What are the ingredients necessary for PCR?

A
  • template DNA
  • 2x ssDNA oligonucleotide primers
  • 4x dNTPs
  • taq polymerase
  • thermal cycler
67
Q

From where is taq polymerase derived?

A

Thermus aquaticus

68
Q

Describe PCR

A
  • 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
Q

How long does library screening take?

A

several weeks

Hilary Term 1 (61 decks)

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