Introduction to Molecular Genetics

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Introduction to Molecular Genetics Lectures
4-9 Edgar Hartsuiker Genome Damage and
Stability Centre e.hartsuiker_at_sussex.ac.uk
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  lectures 4-9
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• Learning outcomes Lecture 4
• you should be able to understand
• the basic steps of cloning
• the features of restriction endonucleases
• the principle of DNA ligation
• the elements that make up a good vector
• the principle of insertional activation of LacZ'
  and the use of blue/white screening

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Purpose of cloning To amplify or express a
single piece of DNA (gene) in a host organism
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E. coli biology provides the basic tools that are
needed for cloning plasmids restriction
enzymes bacteriophage DNA ligases Over the
years these tools have been adapted to increase
ease of cloning
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Basic steps of cloning
cut ligate
E. coli

Vector
transform

Insert DNA
Replication
extract and purify
Bacterial division
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Double strand DNA nucleases
-A-C-G-A-A-T-T-C-C-A
-T-G-C-T-T-A-A-G-G-T

• Exonuclease
• Cleaves phosphodiester bond of terminal
  nucleotide
• Not suitable for cloning

-A-C-G-A-A-T -T-G-C-T-T-A
T
A
G
A
C
G
T
C
-A-C-G-A-A-T-T-C-C-A
-T-G-C-T-T-A-A-G-G-T

• Endonuclease
• Cleaves phosphodiester bond of internal
  nucleotide
• Suitable for cloning

-A-C-G-A-A -T-G-C-T-T
T-T-C-C-A A-A-G-G-T
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• Specific and non-specific endonucleases
• Non-specific endonuclease
• cuts at random position
• not suitable for cloning
• Specific endonuclease
• cuts at specific recognition sequence
• suitable for cloning

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Restriction Endonucleases play a central role in
DNA cloning strategies
cut DNA to be inserted
cut vector
Restriction endonuclease
Ligation
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Wide range of Restriction Endonucleases
commercially available Isolated from a wide
range of bacteria
Enzyme Recognition sequence Sticky or
blunt EcoRI GAATTC Sticky BamHI GGATCC
Sticky HindIII AAGCTT Sticky PvuII CAGC
TG Blunt Sau3A GATC Sticky
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Restriction endonuclease recognition sequences
are often palindromic In language, a palindrome
reads the same forwards and backwards, e.g.
kayak Different meaning in molecular biology
a sequence equal to the complementary strand read
backwards, e.g. GAATTC

T-T-C-
-G-A-A
-N-N-G-A-A-T-T-C-N-N-
-N-N-C-T-T-A-A-G-N-N-
-N-N N-N- -N-N N-N-
-G-A-A
T-T-C-

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Length of recognition sequence determines cutting
frequency When DNA sequence would be
random length of recognition sequence Cuts
once every 4 44256 bp 6 464096
bp 8 4865536 bp But DNA sequence is
not random RE Predicted cuts in
?-DNA Observed cuts BglII 12 6 BamHI
12 5 SalI 12 2
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Restriction endonucleases can create blunt or
cohesive DNA ends Restriction enzymes cleave
phosphodiester backbone
-N-N-A-G-C-T-N-N-
-N-N-T-C-G-A-N-N-
-N-N-G-A-A-T-T-C-N-N-
-N-N-C-T-T-A-A-G-N-N-
AluI
EcoRI
-N-N-A-G -N-N-T-C
C-T-N-N- G-A-N-N-
-N-N-G -N-N-C-T-T-A-A
A-A-T-T-C-N-N- G-N-N-
Blunt ends
Cohesive ends
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Ligation of DNA fragments by DNA
ligase Joining two DNA molecules together by
creating a phosphodiester bond Opposite of
restriction endonuclease
nick
-N-N-G A-A-T-T-C-N-N-
-N-N-C-T-T-A-A G-N-N-
-N-N-A-G -N-N-T-C
C-T-N-N- G-A-N-N-
DNA ligase
DNA ligase
-N-N-G-A-A-T-T-C-N-N-
-N-N-C-T-T-A-A-G-N-N-
-N-N-A-G-C-T-N-N-
-N-N-T-C-G-A-N-N-
Cohesive end Easy to ligate
Blunt end Difficult to ligate
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DNA ends need to be compatible for successful
ligation
G-A-T-C-C-N-N- G-N-N-
-N-N-G A-A-T-T-C-N-N-
-N-N-C-T-T-A-A G-N-N-
-N-N-G -N-N-C-T-T-A-A
Compatible cohesive ends
Non-compatible cohesive ends
-N-N-A-G -N-N-T-C
C-T-N-N- G-A-N-N-
-N-N-G -N-N-C-T-T-A-A
C-T-N-N- G-A-N-N-
Compatible blunt ends
Non-compatible cohesive/blunt ends
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Use of DNA Ligase in cloning
cut vector
Incubate with DNA ligase


Recombinant DNA molecule
cut insert DNA
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• Cloning vectors
• Adapted from naturally occurring plasmids
• Need to have
• Replication origin
• Suitable (unique) restriction recognition
  sequences
• Selectable marker
• Also useful
• Easy selection of recombinants (insertional
  inactivation)
• Promotor for gene expression

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An Early cloning vector pBR322
EcoRI
HindIII
Unique restriction sites
ScaI
BamHI
PvuI
Ampicilline marker (antibiotic resistance)
ampR
Tetracycline marker (antibiotic resistance)
PstI
tetR
SalI
ori
Replication origin
Antibiotic markers can be used for identificatio
n of plasmid containing bacteria
(selection) identification of recombinant vector
molecules (insertional inactivation)
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Identification of recombinant vector molecules
through insertional inactivation Principle
gene on plasmid is disrupted by inserting DNA
insert
ampR
ampR
tetR
tetR
Plasmid containing bacteria will grow
on ampicillin and tetracycline containing media
Plasmid containg bacteria will grow on ampicillin
but not on tetracycline containing media
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Improved vector pUC8 High copy number Multiple
Cloning Site (MCS) Blue/white screening
(insertional inactivation of LacZ')
ampR
HindIII PstI Sal1, AccI,HincII BamHI SmaI,
XmaI EcoRI
lacZ'
Multiple cloning site (MCS) A cluster of
multiple unique restriction sites
ori
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Use of MCS directional cloning Both vector and
insert DNA are digested with two different
restriction enzymes to create non-compatible ends
on either site of DNA fragment Advantages
prevents self ligation of vector allows only
one orientation of insert
Yes!
EcoRI
BamHI
No!
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Blue/White screening background LacZ part of
the LAC operon, encodes for ß-galactosidase,
O
LacZ
P
IPTG
O
LacZ
P
X-gal
ß-galactosidase
Add IPTG and X-gal to wild type E. coli ?
colonies turn blue
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Mechanism of Blue/White screening Special E.
coli strains lack LacZ' fragment (LacZ'-) Only
blue when complemented with LacZ' fragment on
plasmid LacZ' inactivated when plasmid contains
insert ? white colonies!
E. coli chromosome
LacZ'
LacZ' -
Complete ß-galactosidase molecule