1

CMB2000 Lecture 3

Identifying the DNA

Learning outcomes
Describe the technique for isolating DNA
Define what Restriction endonucleases
are and how they are used to cut and
recombine DNA
Explain how PCR works
Use electrophoresis to separate DNA
fragments
Describe genetic transformation
techniques including the use of plasmids
and vectors
Explain the selection techniques

CMB2000 Lecture 3

Identifying the DNA

Uses of plasmids
1) Expression of proteins

CMB2000 Lecture 3

Uses of plasmids
1) Expression of proteins
2) Manipulation of genes
e.g. site-directed mutagenesis

CMB2000 Lecture 3

Uses of plasmids

Probability
a given gene
is present

Number
of clones
ln (1 - P)
N=

ln (1 a/b)

Average insert
size

Total size
of genome

3) Stabilises DNA sequences

4) Making genomic libraries of small genomes
(although plasmids are rarely used for libraries)
5) Maximum insert size ~ 5,000 bases (average 2,000)
Species

Genome size

Number of plasmids
needed

E Coli

4 x 106

800

Human

3 x 109

600,000
600,000
(1.7 million)

CMB2000 Lecture 3

Identifying the DNA

Uses of plasmids
1) Expression of proteins
2) Manipulation of genes
e.g. site-directed mutagenesis
3) Stabilises DNA sequences
4) Making genomic libraries of small genomes
(although plasmids are rarely used for libraries)
5) Maximum insert size ~ 5,000 bases (average 2,000)

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Bacteriophage-

E coli infecting bacteriophages

best studied
Can take larger inserts than
plasmids
20 kb
readily packaged
can infect host cells

Two types of replication:

Lytic pathway - viruses lyse
infected host cell

Lysogenic pathway- virus nucleic

acid incorporates into host cell genome
prophage

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Bacteriophage-

E coli infecting bacteriophages

best studied
Can take larger inserts than
plasmids
20 kb
readily
packaged
-Genome:
total
length 49 kb (length
important
DNA packaging)
can for
infect
host cells

Two types of replication:

Lytic pathway - viruses lyse
infected host cell

Lysogenic~ pathway
- virus
20 kb lysogenic
genes

nucleic acid incorporates into host cell

replace with ~ 20 kb of
genome prophage
inserted foreign DNA

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
Human DNA

Bacteriophage-

CMB2000 Lecture 3

Identifying the DNA

Bacteriophage-
Concatomer

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Bacteriophage-

E coli infecting bacteriophages

best studied
Can take larger inserts than
plasmids
Black = phage DNA;
=20
kbDNA
colour
foreign

readily packaged
In vitro packaging gives a mixture of -phage.
can infect host cells
Each contains a different DNA insert (phage library)

Two types of replication:

Lytic pathway
- viruses
lyse Medium solidifies:
Pour onto
Add molten
Add
phage host cell
infected
agar
library

agar plate

E.coli grow to form

a lawn

Lysogenic pathway- virus nucleic

acid incorporates into host cell genome
prophage
E.Coli in liquid medium

1
0

CMB2000 Lecture 3

Identifying the DNA

Bacteriophage-

1
1

1
2

CMB2000 Lecture 3

Genomic libraries

Cosmids

Looks like a plasmid, but can act like a

phage
Large plasmids - can accept very large
inserts (44kB)
.twice as large as a phage
BUT has COS sites - can be packaged as
a phage (and infect host E.coli)
Also has a gene for ampicillin resistance
- can be screened as a plasmid

1
3

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
Species

Genome
size

20 kbase
insert
(-phage)

44 kbase
insert
(cosmid)

300 kbase
insert
(BAC)

E.coli

4 x 106

600

270

38

Yeast

7 x 107

10,500

4,800

695

Tomato

7 x 108

105,000

48,000

6,950

Human

3 x 109

450,000

205,000

30,000

1
4

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Artificial Chromosomes

BAC = Bacterial Artificial Chromosomes

YAC = Yeast Artificial Chromosomes

lacz

Chloramphenicol

ori

multiple cloning
site
(HindIII, BamHI,
SphI)

ori

Modified F-plasmid
F-plasmid
unusual very large plasmid
allows genetic exchange between bacteria
accepts large inserts (up to 300 Kb)
low copy number (1-2 per cell)

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
Electroporator

Artificial chromosomes
Voltage pulse:
Temporarily disrupts membranes
(pores formed)

Electric potential across membrane

rises by ~ 0.5-1.0 V
Charged molecules (DNA) driven
across pores (electrophoresis)

1
5

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
= a collection of DNA fragments of one
organism, each carried by a plasmid
/virus and cloned in an appropriate host
Need vectors that can hold large inserts
Need a DNA probe to locate a specific
DNA sequences in the library
Used to be the only way to investigate a
genome - now other techniques (L05/L06) but
DNA libraries still very important for
investigating gene function

1
6

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Bacteriophage-

1
7

1
8

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
123

kb ladder
Vector
(no insert)

3
Vector
(with insert)

GGATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
AAGGT

1
9

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries
123
Paper Towels

Gel

Buffer

Membrane

Membrane

2
0

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Southern blotting
123

Ed Southern
Edinburgh University1970s

Membrane

2
1

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

Southern blotting
123

Addition of blocking
reagent
Prevents non-specific
binding
HYBRIDISATION Probe
addition
Probe
only binds to
complementary DNA
STRINGENCY
Probe only remains
WASHES
hybridised to
DNA
complementary
Amplify the signal
Digoxigenin and antidigoxigenin

Membrane

2
2

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

cDNA
DNA libraries

Eukaryotes
DNA

pre-mRNA

Gene interrupted:
exons; coding
introns; non-coding

Transcription

Processing

mRNA
(mature)

Protein

Translation

(A)n
Processing: introns removed, exons joined together, poly(A) tail added to 3-end

Note: 1) DNA is present in all cells at all times

2) A particular mRNA may only be expressed in
certain conditions - the TRANSCRIPTOME

2
3

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

cDNA
DNA libraries

Eukaryotes
DNA

pre-mRNA

Gene interrupted:
exons; coding
introns; non-coding

Transcription

Processing

mRNA
(mature)

Translation

(A)n
Processing: introns removed, exons joined together, poly(A) tail added to 3-end

add ribonuclease inhibitors

Protein

2
4

CMB2000 Lecture 3

wash away unwanted fraction

Eukaryotes
DNA

pre-mRNA

Gene interrupted:
exons; coding
introns; non-coding

Eukaryotes

Transcription

mRNA
(mature)

Processing

elute off purified mRNA

Protein

Translation

(A)n

Processing: introns removed, exons joined together, poly(A) tail added to 3-end

NA

pre-mRNA

Gene interrupted:
exons; coding
introns; non-coding

Transcription

Processing

mRNA
(mature)

Translation

Protein

CMB2000 Lecture 3

Identifying the DNA

Genomic libraries

cDNA
DNA libraries

2
5

CMB2000 Lecture 3

Changed the way nature works

Genomic libraries

cDNA
DNA libraries

Method to clone human insulin in E coli

2
6