BB211: Cell and Molecular Biology

Dr Eve Lutz
Department of Bioscience

Recombinant DNA technology:

Lecture 1
Introduction to Recombinant DNA
technology
Restriction enzymes and gel
electrophoresis
Background reading:

Chapter 10 pp 205-206; Chapter 16

Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

Please see the ReBase website for information regarding

Restriction Enzymes: http://rebase.neb.com/rebase/rebhelp.html

For Restriction Mapping, click on this link: Restriction Mapping

Page
For Additional Help, click on this link: Helpful Hints

An organism's genome contains

virtually ALL the information
necessary for its growth and
development
Determining the molecular sequence of DNA that makes up the
genome of different organisms is an international scientific goal,
several laboratories are participating worldwide in this task
(including the Wellcome Trust Sanger Institute and the Roslin
Institute here in Britain). It is thought that having access to the
complete DNA sequence of an organism can help us not only to
decipher its biology but also help us understand major biological
questions, for instance, what makes one species pathogenic
whereas a related species is not. Or what are the genetic
mechanisms which lead to disease and can they be reversed,
halted or even prevented? It has the potential to help us
understand very complex biological processes which are
dependent on the interaction of a number of different genes (like
development or the transmission and progression of particular
diseases) and which have multifactoral causes and effects. And of
course, in the day to day world, we use particular gene products
(proteins and enzymes, peptides) that have been 'adapted' for
commercial use (therapeutics, laundry detergents, genetically
modified organisms such as tomatoes, rice, sheep etc etc).

How do we obtain DNA and how do

we manipulate DNA?
Quite straightforward to isolate DNA
For instance, to isolate genomic DNA

1. Remove tissue from organism

2. Homogenize in lysis buffer containing guanidine thiocyanate
(denatures proteins)
3. Mix with phenol/chloroform - removes proteins
4. Keep aqueous phase (contains DNA)
5. Add alcohol (ethanol or isopropanol) to precipitate DNA
from solution
6. Collect DNA pellet by centrifugation
7. Dry DNA pellet and resuspend in buffer
8. Store at 4°C
Each cell (with a few exceptions) carries a copy of the DNA
sequences which make up the organism's genome. However,
many genomes are large and complex (for instance the human
genome is made up of ~3000 x 106 base pairs). A particular DNA
sequence (for instance the allele of a gene) can be very small in
comparison. And it probably occurs only once or twice within the
genome (i.e. only one or two copies per cell). This means that a
particular DNA sequence will be present as only a (very) small
part within the complex mixture of DNA sequences that make up
the genomic DNA of that organism.

It is often necessary to 'break up' large DNA molecules into

smaller, more manageable fragments - often to sizes ranging from
100 bp to 2 kb (bear in mind that each resulting DNA fragment is
an individual molecule). These smaller fragments can then be
manipulated more easily - to isolate particular DNA fragments, to
characterize their molecular sequence, to determine their
function, to determine their position in relation to other
sequences within the genome, to use them to express proteins,
etc . . .

How do we manipulate DNA?

It used to be difficult to isolate enough of a particular DNA
sequence to carry out further manipulation and/or
characterization of its molecular sequence. DNA is a
macromolecule - it is made up of a sequence of lots and lots of
deoxyribonucleotides. Large DNA molecules can be fragmented
using 'shearing' forces, in other words mechanical stress to
'shred it', thus creating smaller fragments. However, the
resulting fragmentation is not reproducible - the breakage points
can occur anywhere within the molecule, thus each DNA molecule
will be randomly broken down and various different-sized
fragments can be generated, any of which can have the DNA
sequence of interest. A further difficulty in isolating a particular
DNA fragment is that standard chemical/biochemical methods
are not sufficient to distinguish any part of the genome from
another (after all one DNA molecule is chemically similar to
another).

Progress in understanding genetic mechanisms at the molecular

level was slow. Then came the discovery of various bacterial and
viral enzymes which modify and synthesize nucleic acids (DNA
and RNA), along with the means to produce more outwith the
organism from which they were originally isolated. The application
of these enzymes for manipulating DNA (no matter what the
source) led to the creation of Recombinant DNA Technology
which has enabled great scientific advances in the field of
biology, has created new scientific disciplines and has
revolutionized our world.

Recombinant DNA Technology

Techniques for
- Isolation
- Digestion
- Fractionation
- Purification of the TARGET fragment
- Cloning into vectors
- Transformation of host cell and selection
- Replication
- Analysis
- Expression of DNA

DNA is manipulated using various enzymes that

modify and/or synthesize it
Until 1970 there were no convenient methods available for cutting
DNA into discrete, manageable fragments.

1970 - The Beginning of the Revolution

Discovery of a restriction enzyme in the bacterium Haemophilus
influenzae
 Restriction enzymes
Enzymes that can cut (hydrolyse) DNA duplex at specific sites.
Current DNA technology is totally dependent on restriction
enzymes.

Restriction enzymes are endonucleases

• Bacterial enzymes
• Different bacterial strains express different restriction
enzymes
• The names of restriction enzymes are derived from the
name of the bacterial strain they are isolated from
• Cut (hydrolyse) DNA into defined and REPRODUCIBLE
fragments
• Basic tools of gene cloning

Names of restriction endonucleases

Titles of restriction enzymes are derived from the first letter of
the genus +
the first two letters of the species of organism from which they
were isolated.

EcoRI - from Escherichia coli

BamHI - from Bacillus amyloliquefaciens
HindIII - from Haemophilus influenzae
PstI - from Providencia stuartii
Sau3AI - from Staphylococcus aureus
AvaI - from Anabaena variabilis

Restriction enzymes recognize a specific short

nucleotide sequence
 This is known as a Restriction Site

The phosphodiester bond is cleaved between

specific bases, one on each DNA strand

The product of each reaction is two double stranded DNA

fragments

Restriction enzymes do not discriminate

between DNA from different organisms
Most restriction enzymes will cut DNA which contains their
recognition sequence, no matter the source of the DNA
Restriction endonucleases are a natural part of
the bacterial defense system
• Part of the restriction/modification system found in many
bacteria
• These enzymes RESTRICT the ability of foreign DNA (such
as bacteriophage DNA) to infect/invade the host bacterial
cell by cutting it up (degrading it)
• The host DNA is MODIFIED by METHYLATION of the
sequences these enzymes recognize
o Methyl groups are added to C or A nucleotides in
order to protect the bacterial host DNA from
degradation by its own enzymes

Fig 7-5b, Lodish et al (4th ed)

Types of restriction enzymes

• Type I Recognise specific sequences·but then track along
DNA (~1000-5000 bases) before cutting one of the strands
 and releasing a number of nucleotides (~75) where the cut
is made. A second molecule of the endonuclease is required
to cut the 2nd strand of the DNA
o e.g. EcoK.
2+
o Require Mg , ATP and SAM (S-adenosyl methionine)
cofactors for function
• Type II Recognise a specific target sequence in DNA, and
then break the DNA (both strands), within or close to, the
recognition site
o e.g. EcoRI
2+
o Usually require Mg
• Type III Intermediate properties between type I and type
II. Break both DNA strands at a defined distance from a
recognition site
o e.g. HgaI
2+
o Require Mg and ATP

Hundreds of restriction enzymes have been

isolated and characterized
• Enables DNA to be cut into discrete, manageable fragments
• Type II enzymes are those used in the vast majority of
molecular biology techniques
• Many are now commercially available

Each restriction enzyme will recognize its own

particular site
• Some recognize very short sequences consisting of only 4
base pairs. These tend to cut DNA more frequently
(generating smaller fragments) as the likelihood that any
stretch of DNA sequence will contain these minimal
recognition sites is high.

i.e. approximately 1 site per 256 bases ([1/4]4)

 • Some require longer recognition sequences (up to 8 bp). The
longer the recognition sequence the less frequently these
sites are likely to occur in any particular DNA sequence.
Enzymes which cut DNA very infrequently are known as
RARE cutters.

i.e. an 8 bp recognition site will occur approximately 1 per 65,536

bases ([1/4]8)

The sites occur more randomly than predicted, so that digestion

by any one enzyme will generate DNA fragments of different
lengths

Some recognize more than one sequence

• There are restriction enzymes which allow substitutions in
one or more positions of their recognition sequences.
• Most common substitutions
o purines (A or G), designated R
o pyrimidines (C or T), designated Y
o any nucleotide, designated N

For example HincII will allow two substitutions in each of two

sites. It recognizes and cuts 4 different sequences.

5'-G T C GA C-3' 5'-G T T G A C-3' 5'-G T C A A C-3'

5'-G T T A A C-3'
3'-C A G C T G-5' 3'-C A A C T G-5' 3'-C A G T T G-5'
3'-C A A T T G-5'

The consensus HincII recognition site is designated 5'-G T Y R A

C-3'

Many Type II restriction endonuclease

recognize PALINDROMIC sequences
 • Symmetrical sequences which read in the same order of
nucleotide bases on each strand of DNA (always read 5' 3')

For example, EcoRI recognises the sequence

5'-G A A T T C-3'

3'-C T T A A G-5'

The high specificity for their recognition site

means that DNA will be cut reproducibly into
defined fragments
• Generate restriction maps
• Isolate and clone specific DNA fragments

Different enzymes cut at different positions and can create

single stranded ends ('sticky ends')

• Some generate 5' overhangs - eg: EcoRI

• Some generate 3' overhangs - eg: PstI

• Some generate blunt ends - eg: SmaI

 Examples of restriction enzymes and the
sequences they cleave

Source Recognition Ends

Enzyme
microorganism Site produced
Arthrobacter luteus Alu I AG¬ CT Blunt
Bacillus amyloiquefaciens
Bam HI G¬ GATCC Sticky
H
Escherichia coli Eco RI G¬ AATTC Sticky
Haemophilus gallinarum Hga I GACGC(N)5¬ Sticky
Haemophilus infulenzae Hind III A¬ AGCTT Sticky
Providencia stuartii 164 Pst I CTGCA¬ G Sticky
Nocardia otitiscaviaruns Not I GC¬ GGCCGC Sticky
Staphylococcus aureus
Sau 3A ¬ GATC Sticky
3A
Serratia marcesans Sma I CCC¬ GGG Blunt
Thermus aquaticus Taq I T¬ CGA Sticky

The 'sticky' overhangs are known as

COHESIVE ENDS
• The single stranded termini (or ends) can base pair
(ANNEAL) with any complementary single stranded termini

This is the basis for RECOMBINANT DNA

TECHNOLOGY
• Inserting foreign DNA into a cloning vector

Restriction enzymes are a useful tool for

analyzing Recombinant DNA
After ligating a particular DNA sequence into a cloning vector, it
is necessary to check that the correct fragment has been taken
up. Sometimes it is also necessary to ensure that the foreign
DNA sequence is in a certain orientation relative to sequences
present in the cloning vector.

• Checking the size of the insert

• Checking the orientation of the insert
• Determining pattern of restriction sites within insert DNA

DNA fractionation
Separation of DNA fragments in order to isolate and analyze
DNA cut by restriction enzymes

Electrophoresis
Linear DNA fragments of different sizes are resolved according
to their size through gels made of polymeric materials such as
polyacrylamide and agarose. For instance, agarose is a
polysaccharide derived from seaweed - and gels formed from
between 0.5% to 2% (mass/volume i.e. 0.5 to 2.0g agarose/100 ml
of aqueous buffer) can be used to separate (resolve) most sizes
of DNA

DNA is electrophoresed through the agarose gel from the

cathode (negative) to the anode (positive) when a voltage is
applied, due to the net negative charge carried on DNA
When the DNA has been electrophoresed, the gel is stained in a
solution containing the chemical ethidium bromide. This compound
binds tightly to DNA (DNA chelator) and fluoresces strongly
under UV light - allowing the visualization and detection of the
DNA.

Other useful DNA modification enzymes used for

manipulating DNA:

Alkaline Removes phosphate groups from 5' ends of DNA

phosphatase (prevents unwanted re-ligation of cut DNA)
Joins compatible ends of DNA fragments (blunt/blunt
DNA ligase
or complementary cohesive ends). Uses ATP
Synthesizes DNA complementary to a DNA template in
DNA polymerase
the 5'-to-3'direction. Starts from an oligonucleotide
I
primer with a 3' OH end
Digests nucleotides progressively from a DNA strand in
Exonuclease III
the 3' -to-5' direction
Polynucleotide Adds a phosphate group to the 5' end of double- or
kinase single-stranded DNA or RNA. Uses ATP
RNase A Nuclease which digests RNA, not DNA
Taq DNA Heat-stable DNA polymerase isolated from a
polymerase thermostable microbe (Thermus aquaticus)