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GENETICS 2018
Gene cloning – construction and screening of DNA libraries ✓✓
At the end of this lecture you should be able to:

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Understand the components of a DNA library
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A DNA library represents all of the genes in a given tissue or cell etc
...
The DNA is not naked, as it has to be stable
It is genomic if it is all of the genes from an organism, or a cDNA library if it only
represents the genes expressed
These libraries are usually less than 200µl (would normally contain about 2x109 clones, but
1x106 clones - about 0
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Pros:
Small size so have less shearing and are easier to manipulate
Large variety available so easy to find the right restriction site
Many useful features (multiple cloning sites, antibiotic selection…)
Cons
 Upper limit of insert DNA is about 10kb so would need a lot for the full genome
 Normally only use cDNA (as inserts are smaller)
Bacteriophage lambda:
A natural enemy of bacteria, mid-range size of 15-20kb, readily available
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The middle section of the viral genome is replaced with
a linear plasmid, containing multiple cloning sites
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High capacity vectors

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Vector

Type

Cosmid
P1
PAC

like a circular λ
Phage based
Phage based

BAC – bacteria
artificial
chromosome
YAC – yeast
artificial
chromosome

Cell thinks it’s
its own
chromosome
Cell thinks it’s
its own
chromosome

Capacity
(kb)
30-45
70-100
130-150

Host
E
...
coli
E
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high
1
1

120-300

E
...
A
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Look and act like a
normal chromosome
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Only has one restriction site
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coli plasmid, with many cloning sites
 P1 artificial chromosomes have
multiple antibiotic selection markers
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coli (so they kill
Cosmid
themselves off)
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Has multiple selection sites
o Useful vector features include: a multiple cloning site (MCS) for a few to many unique
restriction endonuclease sites, a ribosome binding site (shine-dalgamo for E
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The oligo(dT) adaptor allows a primer to anneal to the tail, the primer automatically
including a Xho1 restriction site, allowing RNA reverse transcription
RNase H degrades most of the RNA leaving only bits, which then work as primers to
transcribe the whole DNA sequence
T4 PNK phosphorylates the 5’ ends, for ligation

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Adding EcoRI linkers adds
restriction sites for both ends,
and the 3’ one is removed to
leave sticky ends for ligation into
lambda phage
 cDNA library derived from mRNA
(which isn’t suitable as it is too
easily degraded)
 Phage lambda normally used as it
has lytic abilities, but others may
be used
Genomic library construction breaks
down into three steps:
1 Isolation of high quality
genomic DNA
2 Preparation of a compatible
vector
3 Insertion of genomic DNA into
the vector
 The isolated genomic DNA is cut
with the same enzyme as the
cosmid
 They are then ligated together,
and recircularised
 Vector is usually phage lambda,
or high capacity vectors

Describe the basis of library screening

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When screening for clones you have to make sure there is complete
coverage
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For genomic libraries this means calculating the probability that a given
sequence is represented, and using that, the size of your insert, and the size of the
total genome, you can calculate the exact number of
clones needed
o
For cDNA libraries it is guesswork – but for
both it’s normally around a million
o
A common method of screening is via
radiolabelled specific nucleic acid probes (bits of
ssDNA which code for the sequence you want)
...
coli,
which contain naked recombinant DNA (with lots of
different genome fragments)
...
Then X-ray to locate the plate and
plaque with the fragment, and isolate the DNA
...
The detection method for the protein of interest must be sensitive and
robust, eg electrophysiology or FACS

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PCR ✓✓
At the end of this lecture you should be able to:

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Understand the components of a PCR
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When it was invented in the 1980s it used normal DNA polymerases which had to keep
being replaced after each cycle
...
Proofreading thermostable polymerases were discovered and boosted PCR
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The DNA is denatured by heating and small complementary DNA primers
are added
...

There are four stages: Denaturation, Primer annealing, Primer extension, Denaturation
These are repeated up to 40 times, which would give 240 molecules: around a trillion
copies of the original amplicon
As the number of cycles increase, the primer becomes more and more incorporated, with
the third cycle being the first to contain the desired product
Primers may also be called oligonucleotides – polynucleotide molecules which are still
small, not containing a large number of nucleotides
PCR components:
 A thermostable DNA polymerase
 Synthetic oligonucleotide primer (bought in)
 Deoxynucleotide triphosphates (dNTPs – for A,T,C and G)
 Divalent cations (for polymerase active site stabilisation)
 Monovalent cations (to adjust salts etc)
 Buffer
 Template DNA
 A thermocycler

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Thermostable DNA polymerases have two main categories: thermophilic (nonproofreading) and hyperthermophillic (proofreading)
Taq is the most famous
...
It has a two domain structure, with
the N-terminal containing 5’→3’ exonuclease activity, and the C-terminal has a DNA
polymerase domain (and an inactive 3’→5’ proofreading exonuclease domain – why it is
less accurate)
...
It has a relatively
fast processivity speed of 42 (2kb/min), but a high error rate
...
The main difference is the
presence of 3’→5’ exonuclease activity, meaning that it can correct mistakes
...
The products are blunt ended (as the A is
removed), and have a very low error rate
...

Nucleotides must be deoxygenated ones (dNTPs) and have to be at a concentration of
more than 50µM for Taq, and 200µM for proofreaders like Pfu
M2+/Mg2+ (free divalent cations) are needed by all thermostable DNA polymerases
...
5-2mM Mg2+ is normally used)
Manganese can be used instead of magnesium, but it makes polymerases very inaccurate
– can be good to randomly induce mutations to study gene function
The template must be DNA, and normally aim for around 3x105 bits of target sequence
...
G≡C rich templates are hard to denature so
can cause issues
...
3-8
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Kept as short as possible - usually 3 mins
2 Primer annealing - ideally 3 - 5°C lower than Tm, usually only 15 - 30 secs
3 Extension – at optimal temp
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The
time depends on processivity of enzyme
4 Denaturation - 94°C for 15 secs
5 A number of cycles (typically 25 - 40)
6 Final extension – at the same temperature as 3, for about 10 minutes
The reaction plateaus due the primers degrading, and running out of dNTPs
Hotstart PCR – can increase specificity - the polymerase activity is delayed until the
reactions are at temperature
...
These have their
active sites filled with a denaturable antibody
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Initial temperature is set 3oC above the Tm of the
primers, and is decreased by 1oC per pair of cycles
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They must be designed to be very specific, and only to
the flanking sequences of the amplicon
They are normally bigger than 18 bases (to stabilise) but less that 30 (or self-anneals),
avoid polymeric tracts, avoid regions which form obvious secondary structures and
inverted repeats, have a G≡C content of at about 50%
...
Taq produces Atailed DNA, whereas Pfu (etc) make blunt ended DNA
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Isolate mRNA, reverse transcribe with
oligo(dT) or (pdN)6 (strings of 6 nucleotides with all possible combinations) primers, then
amplify ds cDNA from ss cDNA using specific primers
RACE – Rapid Amplification of cDNA Ends – recreates the missing ends of cDNA clones
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 Purify plasmid DNA template from a dam+/dcm+ strain of
E
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coli and screen for the mutation
Inverse PCR – clones an unknown sequence which
flanks a known sequence
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Used to give an idea of the
change in gene expression
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The main method is the enzymatic method of Sanger, but there is also the chemical
method of Maxim and Gilbert (which is good for epigenetics and protein binding)
...
It incorporates radiolabelled
DNA as a detection method, and templates can be ssDNA,
dsDNA, dsDNA plasmids, PCR products etc (basically DNA)
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 A

synthetic primer is annealed
to the template (need to know
G
C
T
some of the sequence – or can
generate like with reverse
PCR)
 Four reactions each
containing all the dNTPs and
DNA polymerase, each with
one of the four normal bases
labelled
 Complementary ddNTPs to
the labelled normal base are
added to each reaction (eg
ddTTP to the A reaction etc)
 ddNTP incorporation leads
to chain termination
o Maxim-Gilbert chemical sequencing was developed earlier and was more popular, but it
relies on pairing the bases with several dangerous chemicals:
 Formic acid (purine specific – adenine and guanine)
 Dimethyl sulphate (guanine only)
 Hydrazine (pyrimidines - cytosine and thymine)
 Hydrazine with NaCl (cytosine only)
o Modified DNAs were then cleaved by hot piperidine at the modified
base positions
o The modification reactions were set statistically to modify one base per
molecule
o DNA fragments needed to be less than 300bp, and were normally
prepared from digesting the template (normally end-labelled by T4 PNK,
phosphorylating the 5’ end)
o Labelled dsDNA species denatured, and strands were separated by
denaturing PAGE
o Lots of chemical options to change the cleavage
o Nowadays it is rarely used, except for mapping protein-DNA binding sites – end label the
DNA that you think the protein will bind to, and methylate all molecules once
...
ddNTPs are
normally fluorolabelled, which allows all samples to be analysed in the same lane with
different coloured fluorescence
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Each peak is a different nucleotide, which the computer analyses for you
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Ribozymes (RNA which acts as an enzyme)
can cut DNA, as a primitive form of todays
enzymes
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Principal types of RNAs produced in cells and their roles:
mRNA: messenger RNA
rRNA: ribosomal, form basic structure of ribosome
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Adaptors between mRNA and amino acids
snRNA: small nuclear
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Processing and chemical modifications of rRNAs
miRNA: micro
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Direct degradation of selective mRNAs
piRNA: piwi-interacting
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Regulate diverse cell processes, sometimes acting as scaffolds
o These can have various structures, and have important functions

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tRNA molecules match amino acids to codons in mRNA for translation
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The whole
structure is modified to generate specificity, not just the anti-codon
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Cap-mediated initiation:
 Begins with a special tRNA (for aa methionine) that can recognize the AUG codon
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 The small ribosomal subunit binds to the 5’ end of mRNA, by its cap and moves
forward (5’ to 3’) in search of AUG codon
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eIF2 consists of three subunits - a, b and g
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 In an active complex the g subunit is bound to GTP and in translation initiation the
GTP is hydrolyzed
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 Phosphorylation on the a subunit reduces dissociation rate of eIF2B, so it collects
and blocks the GDP-GTP exchange
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Phosphorylation of 4E-BPs releases them from eIF4E, allowing
interaction with eIF4G and translation
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RNA form the catalytic centre, with the proteins on the
outside
Ribosomes are catalytic complexes made from more than 50
different proteins and several RNA molecules
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The aminoacyl-tRNA
in the A-site functions as the acceptor for the
growing protein during peptide bond formation
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
E-site: the ribosomal site harbouring decylated tRNA on
exiting the ribosome
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The left diagram shows elongation, where
GTP helps to load the incoming tRNA
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The below diagram shows proofreading
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The right diagram shows termination

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Even small molecule changes can affect the structure and regulation
of mRNA, causing premature stop codons and secondary structures

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Post translational control is mediated by iron
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In active translation elF4G/E and
poly-A-binding proteins protect the transcript form the
degrading deadenylase
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CBC (CAP-binding complex) - First purified on the
basis of affinity with 7mG
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Subunits bind
synergistically to 7mG, without having affinity alone
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Protein complexes including CBC (cap-binding
complex) and the eIF4F (eukaryotic initiation factor 4F) bind to 7mG and recruit the enzymes
and factors to the transcript and mediate further processing, export and translation
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A consensus
sequence within the mRNA allows cleavage at the
3’ end of a gene, and the addition of a poly-A tail
(-200+ adenine residues)
The polyadenylation is an untemplated
modification of a species specific length
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It
can either have directed transport on the
cytoskeleton, random diffusion and trapping (to
translate), or degradation (with local protection
by trapping)
It’s not just protein expression which is important,
but where it is expressed – can change the
properties of the cell
Proteins on the mRNA prevent ribosome assembly and therefore translation (local
regulation)
Zipcode is a 54 nucleotide sequence in the 3’ UTR and is recognized by Zipcode binding
protein-1 (ZBP1), which is required for local translation of b-actin
The UTR can affect where the mRNA is – long 3’ UTR found in cell soma, and short 3’ UTRs
are found in axon cells

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Manipulating gene expression (miRNA, siRNA, shRNA, long non-coding RNA)
At the end of this lecture you should be able to:

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Describe typical non-coding RNA species
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70-90% transcribed – therefore not junk
Can regulate gene expression, guide DNA synthesis or repair
Can be Ribozimes/Riboswitches or RNA-protein complexes
Recruiters, tethers and scaffolds – function in the recruitment of protein factors for the
regulation of chromatin states (used in epigenetic regulation)
Decoys, regulators and Pol II inhibitors – direct effect n the process of transcription,
regulating TF function and localization
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May not all have
functions, but do have functions
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Can occur at all the most important levels (chromatin structure, chromosome
segregation, transcription, RNA processing)
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miRNAs (micro RNAs) – transcribed by RNA pol II so has cap and tail
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Drosha processed pre-miRNA into a hairpin type
structure in canonical processing
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One strand of this is incorporated into the miRISC complex and the other is
degraded
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The miRNAs pair with their targets via a
short 5’ region of their sequence – the seed
region
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Evolution of these clusters likely to have
involved gene duplications
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Clustered
miRNAs seem to evolve more rapidly than individual miRNAs

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