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+
copying & sequencing
DNA
module 2 – biotechnology & gene technologies
+
From the spec
+
learning objectives

Understand what the polymerase chain reaction is.

Understand what the PCR can be used for.

Understand how genes can be sequenced.
+
success criteria

Describe what happens in the polymerase chain reaction.

State what is required for the PCR to occur and explain why
these components are important.

State advantages and disadvantages of the PCR.

Explain how fluorescently tagged nucleotides can be used to
sequence genes.
+ starter

Draw and annotate a diagram to show how DNA replication
occurs.

Include as much detail as you can – enzymes, primers etc…

Volunteer to do it on the board, if you’re brave…
+
part 1
copying DNA
+ polymerase chain reaction (PCR)

PCR is an method by which DNA
can be replicated in the lab.

It can be used to create millions of
copies of DNA in just a few hours.

It is essential in forensic science as
very small samples of DNA are
difficult to analyse.

This process amplifies DNA, so
that it can be analysed.
+ what do you need?
1.
A thermocycler – a machine that can quickly alter the
temperature of the samples being amplified.
2.
RNA primers provide the starting sequence for DNA
replication. They also stop the two DNA strands from
joining together.
3.
DNA nucleotides containing the bases adenine, guanine,
cytosine and thymine.
4.
The enzyme - DNA polymerase.
The Double Stranded DNA
Molecule
A
A G G
T
C
A
C
T
T
T
T
A G
T
G A
A
C C
Heat to 950C to separate the DNA strands
DNA Strand
A
A G G
C
T
T
C
T
A
C
T
C C
T
RNA Primers
C
T
T
A G
T
T
T
G A
A
DNA Strand
Cool to 550C to allow primers to bind (anneal) to
DNA
DNA Polymerase
G
Original DNA strand
A
A G G
T
C
A
T
T
A G
T
Primer
G
C C
G
T
T
C
C
Nucleotides join on
Free DNA
nucleotides
A
A
T
G
C
T
G
Nucleotides join on
G
C C
T
C
A G
A
T
G
CPrimer
T T
G A
A
Free DNA
nucleotides
Original DNA strand
Mix with DNA polymerase and free nucleotides and heat to
720C
+ a cyclic process

The whole process can be repeated many times, so the
amount of DNA increases exponentially.

x2, x4, x8, x16, x32 etc.
Strand Separation
DNA heated at 95°C for
5mins
Mix with Primers
(RNA strands)
C
Binding of Primers
Mixture cooled to
55°C
Mix with
Free Nucleotides
DNA Polymerase
REPEAT
CYCLING
With every cycle the amount
of DNA doubles
DNA Synthesis
Mixture heated to
72°C
(optimum temp. for
DNA polymerase)
Suggest why
DNA polymerase
is referred to as
a thermophilic
enzyme.
+ Advantages & Disadvantages

It is a very rapid process –
each cycle takes only
minutes.

It does not require living
cells – only viable DNA
fragments.

It is useful when needing
large amounts of DNA for
genetic engineering.

Any contaminant DNA will
also be amplified.

If a desired gene is to be
copied, any ‘junk DNA’
either side will also be
amplified.
+
part 2
sequencing DNA
+ Meet Frederick Sanger...
 Biochemist
 Cambridge
University
 English
 Two
 Still
Nobel Prizes
Alive
Sanger’s work in the 1970’s, which
earned him his second Nobel Prize,
involved the sequencing of DNA.
Sanger Sequencing Method
His method used modified nucleotides that do not
allow another nucleotide to join after them in a
+ Introducing Sanger Sequencing
 The
method is based on the premature ending of DNA
synthesis.
 If
modified nucleotides are used during DNA synthesis, the
process can be halted.
What normally happens during DNA synthesis...
T A T G G A T C T G A C C T T A G
A T A C C T A G A C T G G A A T C
What happens if you modify a nucleotide...
You call these modified
nucleotides,
T A T G G A T C
TERMINATORS
A T A C C T A G A C T G G A A T C
+ what you need for sequencing

The reaction mixture for automated sequencing is very similar
to that of PCR.

However, some of the nucleotides are fluorescently labelled,
and if added to a growing chain, no further bases can be added.
What you need:
A
T
The DNA being sequenced.
A mixture of ‘normal’ nucleotides (A, T, C, D)
A primer.
DNA Polymerase.
Fluorescently labelled ‘terminator’ nucleotides.
G
C
A
C
C
So what happens in each tube?
+
 Lets take the example of the tube with an adenine
terminator Now let’s imagine this is the sequence of the
unknown DNA strand:
A
C C GT CT A G C A CT C A A G CT C
T
T
What are the possible
terminated sequences going to
be when the reaction is over?
G
A
A
C
C
Because there are both ‘normal’ and
GGCA
‘terminator’ nucleotides in the mixture,
G G C A G A there is a chance that either is placed
as the next base
G G C A G AT C GT G A
G G C A G AT C GT G A GTT C
G
GA
G C A G AT C GT G A GTT C
+ Remember that this is happening in four
test-tubes, each with a different type of
terminator nucleotide.
DNA fragments in each of the four tubes are
going to be of varying lengths.
Now the lengths of DNA need to be
separated, so that we can see why we
went through all of this trouble...
Back
to
Sanger
Sequencing
 The fragments produced during the reactions can be separated using
gel electrophoresis.

The smallest fragments will move furthest along the gel in a fixed
period of time.

Due to being radioactively labelled, we can see where the DNA
fragments end up, by placing photographic film over the gel, after the
run.
Terminat
or C
Terminat
or A
Terminat
or T
Terminat
or G
Modern Sequencing - what the fluorescent bases
do

If a modified (terminator) nucleotide is added, the DNA
polymerase is ‘thrown off’ and can no longer be completed.

It will be shorter than a fully replicated molecule.
What normally happens during DNA synthesis...
T
A
T
G
G
A
T
C
T
G
A
C
C
T
T
A
G
A
T
A
C
C
T
A
G
A
C
T
G
G
A
A
T
C
What happens if you modify a nucleotide...
T
A
T
G
G
A
T
C
A
T
A
C
C
T
A
G
Remember that these modified
nucleotides are fluorescently labelled.
A
C
T
G
G
A
A
T
C
+ 4 bases, 4 colours

As there are 4 different bases, each type of modified
nucleotide has to have an individual fluorescent marker.
Adenine

Thymine
Cytosine
Guanine
As the reaction proceeds (and modified nucleotides halt
individual reactions), varying lengths of DNA are made:
Molecules of every
T A GT C A G
possible lengths
T A GT C A GT A AT
would be made…
T A GT C A GT A AT A G CT AT
T A GT C A GT A AT A G CT AT A C
T A GT C A GT A AT A G CT AT A C G AT A GT
T A GT C A GT A AT A G CT AT AT G AT A GT AT A A
T A GT C A GT A AT A G CT AT AT G AT A GT AT A AT A
+ spot the colour

The strands are separated according to size in a process
similar to gel electrophoresis.
A computer analyses the order of fluorescent markers,
and can therefore deduce the order of bases in the
template DNA strand.
Using the key below, work out the order of bases on the
template DNA strand:
A
T
C
G
+
Plenary
1.
Explain how the PCR enables forensic scientists to analyse
minute samples of DNA found at the scenes of crime.
2.
Suggest why DNA polymerase used in the PCR is obtained
from thermophilic bacteria.
Homework

Stretch and Challenge section on page 171 of textbook.
+
learning objectives

Understand what the polymerase chain reaction is.

Understand what the PCR can be used for.

Understand how genes can be sequenced.
+
success criteria

Describe what happens in the polymerase chain reaction.

State what is required for the PCR to occur and explain why
these components are important.

State advantages and disadvantages of the PCR.

Explain how fluorescently tagged nucleotides can be used to
sequence genes.
+

•
Locating and sequencing genes
DNA probes
Using BAC gene library  DNA sequencing
Gel Electrophoresis
•
PCR
+
Flow diagram: Sequencing the
Genome

Use resources (p184-8 green book)

Restriction enzymes

BAC

Growth of bacteria

Extraction and RE

Sequencing

Computer processing