Download Restriction enzymes

Chapter 11
Molecular tools and
techniques
DNA Profiling
• Restriction enzymes
• Gel electrophoresis
• The Southern Blot technique
• DNA amplification- Polymerase Chain Reaction
(PCR)
• Sequencing DNA.
• Gene technology refers to the manipulation and
use of DNA.
• DNA profiling is characterising the genetic makeup
of an individual by cutting DNA into fragments
using restriction enzymes and examining the
banding pattern on an electrophoresis gel.
• Enzymes are important in all gene technologies.
(What a surprise!!)
• The enzymes used have been discovered in, and
extracted from the cells of
• eukaryotes,
• prokaryotes
• and viruses.
Restriction enzymes

Restriction enzymes are made by bacteria to
protect themselves from viruses. They inactivate
any viral DNA by cutting it in specific places.
 If foreign DNA enters the bacteria cell the restriction
enzyme will cut it up into small pieces. A process
known as restriction. They cut up only certain base
pair sequences and thus are handy in genetics.
 Restriction enzymes can be used to cut DNA at
specific sequences called recognition sites. They then
rejoin the cut strands with DNA ligase to form new
combinations of genes.
Restriction Enzymes
Restriction enzymes are one of the essential tools of genetic engineering.
Purified forms of these naturally occurring bacterial enzymes are used as
“molecular scalpels”, allowing genetic engineers to cut up DNA in a
controlled way. Also called endonucleases.
Restriction enzymes are used to cut DNA molecules at very precise
sequences of 4 to 8 base pairs called recognition sites (see below).
By using a ‘tool kit’ of over 400 restriction enzymes recognizing about
100 recognition sites, genetic engineers are able to isolate and
sequence DNA, and manipulate individual genes derived from any type
of organism.
Recognition Site
The restriction
enzyme EcoRI cuts
here
DN
A
Recognition Site
cut
GAATTC
GAATTC
CTTAAG
CTTAAG
cut
cut
Specific Recognition Sites
Restriction enzymes are named according to the bacterial species they were first
isolated from, followed by a number to distinguish different enzymes isolated
from the same organism.
e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.
Restriction enzyme
EcoRI
BamHI
HindIII
Bacterium
Escherichia coli
Strain RY 13
First endonuclease isolated
Bacillus amyloliquefaciens
Strain H
First endonuclease isolated
Haemophilus influenzae
Strain Rd
Third endonuclease isolated
A restriction enzyme cuts the double-stranded DNA molecule at its
specific recognition site:
Enzyme
Source
Recognition Sites
EcoRI
Escherichia coli RY13
GAATTC
BamHI
Bacillus amyloliquefaciens H
GGATCC
HaeIII
Haemophilus aegyptius
GGCC
HindIII
Haemophilus influenzae Rd
AAGCTT
Hpal
Haemophilus parainfluenzae
GTTAAC
HpaII
Haemophilus parainfluenzae
CCGG
MboI
Moraxella bovis
GATC
NotI
Norcardia otitidis-caviarum
GCGGCCGC
TaqI
Thermus aquaticus
TCGA
Cutting specificity
• When DNA is cut with a restriction enzyme the resulting
fragments are either left with a short overhang of single
stranded DNA called a ‘sticky end’ or no overhanging DNA
called a ‘blunt end’.
GAATTC
GTTAAC
CTTAAG
CAATTG
EcoRI – leaves a sticky end.
HpaI – leaves blunt ends
Sticky Ends
It is possible to use
restriction enzymes that
A restriction enzyme cuts the doublestranded DNA molecule at its specific
recognition site
Fragment
cut leaving an overhang;
Restriction
enzyme: EcoRI
GAAT T C
GAAT T C
C T TAA G
C T TAA G
a so-called “sticky end”.
DNA cut in such a way
Restriction enzyme: EcoRI
produces ends which may
only be joined to other
AAT T C
G
sticky ends with a
complementary base
sequence.
G
DNA from
another
source
C T TAA
Sticky end
The cuts produce a
DNA fragment with
two “sticky” ends
The two different fragments
cut by the same restriction
enzyme have identical sticky
ends and are able to join
together
See steps 1-3
opposite:
When two fragments of DNA cut by the same
restriction enzyme come together, they can join by
base-pairing
Blunt Ends
Recognition Site
It is possible to use restriction
enzymes that cut leaving no
Recognition Site
C C CG G G
C C CG G G
G G GC C C
G G GC C C
DNA
overhang; a so-called “blunt
end”.
cut
DNA cut in such a way is able to
be joined to any other blunt
Restriction enzyme
cut
cuts here
The cut by this type of restriction
enzyme leaves no overhang
CCC
end fragment, but tends to be
GGG
CCC
non-specific because there are
no sticky ends as recognition
sites.
A special group of
enzymes can join
the pieces together
GGG
GGG
CCC
CCC
GGG
DNA from another source
Ligation
DNA fragments produced using restriction enzymes may be reassembled by a process called
ligation.
Pieces of DNA are joined together using an enzyme called DNA ligase.
DNA of different origins produced in this way is called recombinant DNA because it is DNA
that has been recombined from different sources.
Steps 1-3 are involved in creating a recombinant DNA plasmid:
Two pieces of DNA
are cut using the
same restriction
enzyme.
Plasmid
DNA
fragment
This other end of the
foreign DNA is
attracted to the
remaining sticky end of
the plasmid.
The two different DNA fragments
are attracted to each other by
weak hydrogen bonds.
AAT T C
G Foreign DNA fragment
G
C T TAA
Gel Electrophoresis
• Is one of the most commonly used tools.
• It is a molecular separating technique used to sort molecules
– DNA and proteins on the basis of size, electric charge, and
other physical properties.
Brief outline:
• DNA is extracted from cells.
• It is cut using restriction
enzymes.
• Added to a gel tank.
• Electric current is run
through it.
• DNA fragments migrate
towards the positive
electrode.
• Smaller fragments move
faster.
DNA samples are
loaded into wells
DNA is stained
using ethidium
bromide or
methylene blue.
Electrical current applied
to the chamber
Polymerase chain reaction
To work with DNA it is necessary to have more than a
few molecules. The polymerase chain reaction (PCR) is
a technique used to amplify (make millions of pure
copies of) a piece of DNA in a test tube (Figure 11.4). It
allows, for example, forensic scientists to amplify the
DNA in traces of blood left at the scene of a crime. It is
also used to amplify a particular gene from a sample of
DNA fragments. Biologists developed the technique of
PCR by studying how DNA is synthesised naturally in
cells.
The technique of (PCR)
• PCR is a chain reaction of DNA replication events. The method uses a
complex mixture of ingredients, which is heated and cooled in cycles.
At each cycle of synthesis, the number of copies of the DNA fragment
is doubled. A large amount of DNA can be produced in a test tube in a
few hours.
• The polymerase chain reaction (PCR) mixture contains four ingredients:
• a sample of DNA, which acts as a template to make millions of copies
• a source of the four nucleotides: A, T, C and G, which are the building
• blocks for DNA replication
• a DNA polymerase (Taq polymerase), which is a heat-resistant enzyme
• single-stranded DNA primers, which are synthetic, short pieces of DNA
• that are complementary to sequences of bases that flank the DNA
region to be amplified. Primers specify the starting and finishing points
for DNA replication (see Chapter 10).
• These ingredients are placed together in a plastic
tube in a DNA thermocycler, a heating block that is
able to change temperature very rapidly. The
thermocycler initially heats to a temperature of
95°C. This breaks the hydrogen bonds and
separates the strands of the DNA sample to make
two single-stranded templates (Figure 11.5). The
thermocycler then cools to a temperature of 50–
60°C to allow the primers to bind to their
complementary DNA sequence. These primers are
typically 18–30 nucleotides in length. The two
primers bind at the ends of the DNA that is to be
amplified; one primer binds to each template
strand.
• The temperature is then increased to approximately
72°C, the optimal temperature for the DNA (Taq)
polymerase enzyme. The enzyme DNA polymerase
begins to move along the template DNA, starting from
the primer and adding nucleotides. Nucleotides are
added at the 3′ end of the new strand according to the
complementary base-pairing rules. Once this first round
of DNA replication is complete, two double-stranded
DNA molecules have been produced from each doublestranded DNA molecule added to the reaction mixture.
The thermocycler then heats to 95°C and the next
cycle of strand separation, binding of primers and DNA
replication begins. Thermocyclers can be programmed
to go through many cycles. Typically, 30–40 cycles are
used to amplify a DNA sample.
• DNA samples can be analysed to explore possible paternal
and maternal relationships between parents and children.
This is possible because half of each person's 23 pairs of
chromosomes come from their mother, and half from their
father.
• Each of the chromosomes contains many sections of noncoding DNA that does not seem to code for a protein, but
contains areas called short tandem repeats (STRs). Each STR
contains repeats of short sequences of bases, such as CATG in
CATGCATGCATG.
• When STRs are tested in DNA profiling, they occur in pairs.
One chromosome in a pair carries an STR from the person's
mother, and the other carries an STR from the person’s father.
• http://www.biotechnologyonline.gov.au/popups/int_dnaprofi
ling.html
• A person’s DNA profile as seen on an electrophoresis gel
usually shows two lines for each of the STRs tested. This is
because usually, the STRs inherited from the parents are of
different lengths. Occasionally, only one line appears because
both STRs in a pair are of the same length.
• When the DNA profile of a child is compared to the profiles of
the child's genetic parents, it is possible to match one line in
each STR area with a line in that area of the mother's profile.
In this way, DNA profiling can also reveal non-paternity.
• Using 10 different STR loci allows for a person to be identified
with a very high level of probability. In America they use 13
STR loci due to their larger population to increase accuracy.
DNA sequencing
• DNA sequencing is used to work out the exact order, or
sequence, of the base pairs in a section of DNA.
• Knowing the base sequence can be helpful if you want to
locate a specific gene by using a gene probe, or to make an
artificial chromosome with a specific gene on it.
• DNA sequencing is also being used to identify and locate all
the genes in an organism. (Eg: Human Genome Project)
• A DNA sequencing machine uses the same principle as
electrophoresis. However, it is so sensitive that it can separate
DNA strands that differ in length by only one nucleotide.
The base sequence of a strand of DNA is worked
out by:
• copying the DNA many times, each time
constructing DNA chains of different lengths
• using electrophoresis to separate the strands
from shortest to longest.
Recombinant DNA technology
• Often called genetic engineering.
• It involves the removal of DNA from one organism to be placed in
another organism of the same or a different species.
• The organism that expresses the foreign genes is called a
transgenic organism.
• This technology requires:
1. a way of extracting or producing the desired gene.
2. a way of making many copies of the DNA.
3. a way of inserting the desired gene into the target organism.
Recombinant DNA Technology
• The major tools of recombinant DNA technology are
bacterial enzymes called restriction enzymes, which
were first discovered in the late 1960s.
• These enzymes protect bacteria against intruding
DNA from other organisms.
• They work by cutting up the foreign DNA, a process
called restriction.
• If foreign DNA enters the bacteria cell the restriction
enzyme will cut it up into small pieces. They cut up
only certain base pair sequences and thus are handy
in genetics
• The bacterial cell protects its own DNA from restriction
by adding methyl groups (--CH3) to adenines or
cytosines within the sequences that would otherwise
be recognized by the restriction enzyme.
PLASMIDS
Plasmids are molecules of
DNA that are found in
bacteria, separate from the
bacterial chromosome.
They:
• are small (a few thousand
base pairs)
• usually carry only one or a
few genes
• are circular
• have a single origin of
replication
Plasmid DNA (a reminder)
Recipient
bacterium
Bacteria have small accessory
chromosomes called
plasmids.
Sex pilus conducts
the plasmid to the
recipient bacterium
Plasmids replicate
independently of the main
chromosome.
Plasmid of the
conjugative type
A plasmid about to
pass one strand of the
DNA into the sex pilus
Plasmid of the
non-conjugative type
Donor
bacterium
Insulin
• Plasmids are able to
transfer from cell to
cell. E coli plasmid is
used to replicate the
insulin gene. This is
then placed in a
bacteria cell and
cultured and insulin
removed.
• By using the same
restriction enzymes
on humans and
bacteria the same
DNA is cut this means
that the gap from the
bacteria DNA can be
filled by the human
insulin gene.
Restriction Enzyme used in insulin
• The insulin gene can
be isolated using
restriction enzyme as
scissors to cut the
insulin gene from the
rest of the human
body.
• We then manufacture
insulin.
• To do this we take the
gene cut by the
restriction enzyme
and put in a plasmid
which are also from
bacterial cells.
Using
Plasmids
Preparation of the Clone
Human cell
Human gene
A gene of interest
(DNA fragment)
is isolated from
human tissue
cells
Restriction enzyme
cuts the plasmid DNA
at its single
recognition sequence,
disrupting the
tetracycline resistance
gene
DNA in
chromosome
Sticky
end
Plasmid
Chromosome
Sticky
end
Restriction enzyme
recognition sequence
Human DNA and
plasmid are treated
with the same
restriction enzyme
to produce identical
sticky ends
Mix the DNAs
together and add
the enzyme DNA
ligase to bond the
sticky ends
Escherichia coli
bacterial cell
Plasmid
vector
Tetracyclineresistance gene
Ampicillinresistance
gene
Gene
disrupted
Sticky
ends
Human gene
Recombinant plasmid is introduced into a bacterial
cell by simply adding the DNA to a bacterial culture
where some bacteria take up the plasmid from
solution
An
appropriate
plasmid
vector is
isolated
from a
bacterial
Recombinant
cell
DNA
molecule
Applications of DNA profiling.
• Establishing parentage.
• Establish other family relationships.
• Analyse biological evidence from a crime
scene.
• Establish the level of genetic variation in
threatened species.
• Establish evolutionary relationships between
groups of organisms.
Application of DNA profiling
• The use of DNA techniques in forensic
science has advantages over other
techniques. This is because DNA is a
stable molecule, which can withstand
drying out.
• The PCR technique allows forensic
scientists to work with very small amounts
of DNA
Application of DNA profiling
• DNA profiling is used to reveal family
relationships in paternity tests or to test
members of a family for a possible inherited
disorder.
• Estimating the amount of genetic variation in
rare and endangered species is useful for
determining a conservation management
strategy.
• Detailed information is provided by gene
sequencing, which has many applications
such as in crop breeding and medicine.
DNA profiling
This is also called DNA
fingerprinting. It uses a
pattern of repeated
DNA sequences (called
‘short tandem repeats’
– STR’s) that are unique
to an individual to
identify a particular
person’s DNA. They are
between genes and are
in the non-coding DNA.
There are more than
10,000 STR loci in one
set of human
chromosomes.
The DNA profile is
observed using gel
electrophoresis
technology.