1

CMB2000 Lecture 1

Isolating DNA
Learning outcomes

Explain how PCR

works

CMB2000 Lecture 2

1. Denature 94C

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1. Denature 94C
2. Anneal by cooling

Hybridisation

3. Extension

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTCTCCCTTCAG
TTGACAGT

Taq DNA polymerase

Kary Mullis (1987)

awarded Noble Prize, 1993

CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1. Denature 94C

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1. Denature 94C
2. Anneal primers

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC

ACCG

AGGG

CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1. Denature 94C
2. Anneal primers

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG
AGGG

CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1. Denature 94C
2. Anneal primers

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG

AGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 1
Copies 2

1. Denature 94C
2. Anneal primers
3. Extension

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG ACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

Taq DNA polymerase

GGATGGCTGCTATTTCCAAAACTGTCAGAGAGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 1
Copies 2

1. Denature 94C
2. Anneal primers
3. Extension

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC

ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGG

CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 1
Copies 2

2
4

1
0

1. Denature 94C
2. Anneal primers
3. Extension

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG ACGATAAAGGTTTTGACAGTCTCTCCC
TGGCTGCTATTTCCAAAACTGTCAGAG AGGG
ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

GATGGCTGCTATTTCCAAAACTGTCAGAGAGGG
ACCG ACGATAAAGGTTTTGACAGTCTCTCCC

TGGCTGCTATTTCCAAAACTGTCAGAGAGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 2
Copies 4

1. Denature 94C
2. Anneal primers
3. Extension

1
1

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 2
Copies 4

3
8

1. Denature 94C
2. Anneal primers
3. Extension

1
2

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 3
Copies 8

4
16

1. Denature 94C
2. Anneal primers
3. Extension

1
3

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Cycles 4
Copies 16

10
1024
30
,
, ,
1073741824

1. Denature 94C
2. Anneal primers
3. Extension

1
4

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Limits
Log amplification phase starts to plateau
Insufficient reagents to complete extension
DNA polymerase makes mistakes
Must be ultra-sterile in your lab skills
Problem sequences

1
5

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Melt map format generated by WAVEMAKER software for the
RFC exon 6 showing base position against the temperature
required to render 75%
in helical
form at to
that plateau
position.
amplification
phase
starts

Log
Insufficient reagents to complete extension
DNA polymerase makes mistakes
Must be ultra-sterile in your lab skills
Problem sequences

1
6

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

1
7

Diagnostics
Agricultural
management

Forensics

PCR
Phylogeny

Basic
research

Drug
development

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Buffer (containing Mg++)
Template DNA
2 primers that flank the fragment of DNA to be
amplified
Four nucleotides (dATP, dGTP, dTTP, dCTP)
Thermostable DNA Polymerase

1
8

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Taq DNA polymerase I from Thermus aquaticus
works optimally at 72oC

Pfu DNA Polymerase from Pyrococcus furiosus

has 3- 5 proofreading activity (more accurate)
works optimally at 75oC

Thermostable DNA Polymerase

5-3 polymerase activity

1
9

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

94C

52C

72C

2
0

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

94C

52C

Thermocyclers

72C

2
1

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Primers
Synthetic oligonucleotides 18-30 bases long
Length only as long as required for specificity
Prevent primer dimer formation
Primer 1 - ACCGTTACGCGGGC
Primer 2
ATGCGCCCGGTTAAG

2
2

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Primers
Synthetic oligonucleotides 18-30 bases long
Length only as long as required for specificity
Prevent primer dimer formation
Prevent self annealing
Absence of hairpins no long self-complimentary
sequences in a primer (< 3 bp):

2
3

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

Primers
Synthetic oligonucleotides 18-30 bases long
Length only as long as required for specificity

2
4

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

2
5

Primers
Synthetic oligonucleotides 18-30 bases long
Length only as long as required for specificity
Annealing temperature must be similar for
each primer in the pair
Ideally have a G or a C at the 3-end
Avoid a T because most common to mismatch
5-CAGTCAACTGCTAC-3 good, but
5-CAGTCAACTGCTGC-3 would be even better

CMB2000 Lecture 2

Polymerase Chain Reaction (PCR)

PCR cloning
GAATTC
CTTAAG

GAATTC
CTTAAG

Adding additional sequences to the ends

that will be recognised by RE
Designing primers which have cut sites
Produces pieces of DNA with sticky ends

2
6

CMB2000 Lecture 2

Isolating DNA
Learning outcomes
Describe genetic transformation
techniques including the use of plasmids
and vectors

2
7

2
8

CMB2000 Lecture 2

GAATTC
CTTAAG

GAATTC
CTTAAG
Cut with EcoRI

G
AATTC
CTTAA
G

G
AATTC
CTTAA
G
DNA ligase
(+ ATP)

G AATTC
CTTAAG
Recombinant DNA molecule

2
9

CMB2000 Lecture 2

AATTC
G

G
CTTAA

DNA to insert
Plasmid vector

EcoRI
G TAA
CT

AATT
C
G

DNA ligase
ATP

Recombinant plasmid

3
0

Transformation

CMB2000 Lecture 2

Plasmids and Vectors

Transformation
heat shock

1. Cold CaCl2

CMB2000 Lecture 2

Plasmids and Vectors

Transformation
heat shock

1. 42C quickly

CMB2000 Lecture 2

Plasmids and Vectors

Vector = any type of plasmid, virus or
artificial chromosome that has been
engineered to accept the insertion of
foreign DNA and can be taken up by living
cells and be replicated, transcribed and
translated.

3
3

CMB2000 Lecture 2

Plasmids and Vectors

Selectable marker
Amp
r

gen
e

pIC19
H
2.7 kb

Origin
of Replication

3
4

CMB2000 Lecture 2

Plasmids and Vectors

Selectable marker
Allows only bacterial (E. coli) cells containing
the plasmid to survive under specific
conditions (presence of antibiotic).
e.g. gene coding for ampicillin resistance

pIC19
H
2.7 kb
Ampr

Origin
of Replication

Plate on
ampicillin

3
5

3
6

CMB2000 Lecture 2

Plasmids and Vectors

Selectable marker
Amp
r
E.coli lacZstrain

Gen
e

X-gal

pIC19
H
2.7 kb

CH2OH
O
HO
O

-galactosidase
-peptide. Inactive

Br
Cl

OH

Truncated -galactosidase
(lacks -peptide). Inactive

pUC

H
N

OH
HO

Non-covalent association
of two inactive components
giving active -galactosidase

CH2OH
O
OH
OH

H
N
Br

HO

OH

Cl

Further reaction to
blue insoluble dye

E coli that grow will

be blue

CMB2000 Lecture 2

Plasmids and Vectors

Amp
r

Gen
e

pIC19
H
2.7 kb
Lac Z
gene

Origin
of Replication

Multiple Cloning
Site
(polylinker)

3
7

3
8

CMB2000 Lecture 2

Plasmids and Vectors

Blue colonies

Amp

Apr r
Ap

Gen plasmid
pIC19H
size
e approx
2700bp

pUC18

LacZ
LacZ

2.7 kb

Lac
Z
Ap
Ap
gene
r

pblt101 recombinant
Origin
size
approx
3200bp
of Replication

LacZ

EcoRI

restriction
enzyme sites in
MCS

White colonies

3
9

CMB2000 Lecture 2

Plasmids and Vectors

Blue / white selection
Amp
r

Gen
e

pIC19H plasmid
size approx
2700bp

Ampr

Blue colonies

Apr r
Ap

Plate on
ampicillin
plus X-gal

pUC18

LacZ
LacZ

2.7 kb

EcoRI

White colonies

DNA insert = recombinant

Lac
Z
Ap
Ap
gene
r

pblt101 recombinant

restriction
enzyme sites in
MCS

Origin
size
approx
All
colonies
are Ampr
of3200bp
Replication
But only some are recombinant
(white ones)
LacZ

Blue/white selection of
recombinant E. coli clones

CMB2000 Lecture 2

-peptide of beta-galactosidase expressed from

plasmid (often termed the LacZ gene)
-peptide expressed from host (E. coli) genome
E. coli containing plasmid without DNA insert
(non-recombinant) metabolise X-gal to a blue
product
Insert in vector disrupts production of betagalactosidase -peptide thus no active enzyme
produced thus colony appears white not blue