3. Polymerase Chain Reaction
(PCR)
The most important discovery in biology since the
discovery of the structure of DNA!
Years ago, when doing analysis of DNA, if DNA
was lost or contaminated it could take months to
make enough to allow a re-analysis!
The discovery of the PCR as a technique to
quickly and accurately create more copies of a
piece of DNA of interest was revolutionary.
4. Summary of the process
The whole process can be summarised in a few
simple steps:
1. Firstly, the DNA template is heated to denature it
into single strands
2. Secondly, the temperature is reduced and primers
SPECIFIC to each of the two denatured DNA
strands are added. The primers anneal to the DNA
strands due to complementary bases being present
3. DNA polymerase (present in the reaction mix)
begins to synthesise new DNA strands
4. You now have two copies of the DNA you started
with and the whole process repeats.
8. Primers and how they function
To begin synthesis of
new DNA, DNA
polymerases need a
region of double
stranded DNA to act
as a template that they
can bind to so as to
begin transcription
from
The primer acts as a
double strand on the
9. Exercise
1. Draw the primer for
the second DNA
template
2. Draw the sequence
of DNA produced
following DNA
synthesis
10. DNA Polymerase
A thermostable enzyme
It can withstand temperatures of 110 oC and still
maintain its functionality
The first PCR DNA polymerase to be used is
called Taq and was isolated from Thermos
aquaticus which was found to live in hot springs
Scientists wondered how it replicated DNA and
was able to live at such high temperatures and
after studying it discovered its SPECIAL DNA
polymerase
11. The need for thermostability
But
polymerase
only begins
to work
here
Polymerase
must
withstand
this
temperature
12. Copies of DNA you can make
After 30 cycles
(which will take
about 2 hours to
carry out) you will
have 536 MILLION
copies of your
starting template
13. PCR uses
PCR has a multitude of uses;
Genetic Testing: to screen for and detect DNA
mutations
Tissue typing: before organ transplant to test for
compatibility
Genetic fingerprinting at crime scenes
Paternity testing
DNA sequencing
DNA cloning
Creating large volumes of DNA for other work
Genetic mapping
17. DNA Probes
A DNA probe is a SHORT length of DNA with
KNOWN nucleotide BASE SEQUENCE
Either has a or labelled
end and so can be used as a marker
18. The uses of DNA probes
The probe will base pair with any complementary
nucleic acid strands
As we know the probe sequence we can use it to
probe and entire GENOME and find any sites of
complementary DNA
21. Detecting minor differences in
DNA
Genetic variation can be caused by either natural
changes in DNA sequence over time, or else by
mutations that spontaneously occur
We can detect very subtle changes in DNA
sequence between individuals with a high degree
of accuracy, caused by genetic variation
22. Genetic Marker Sites
Many diseases can be caused by single
mutations in DNA sequence
This one change can lead to a change in amino
acid in a protein or no protein at all!
Sites that regularly show differences are referred
to as genetic “marker” sites
23. Types of Genetic Marker
There are different types of genetic marker
Restriction fragment length polymorphisms
DNA molecules are cut by restriction nucleases. If
there is a mutation that has occurred at the site of
one of the restriction sites then we will get a
different length DNA fragment following PCR
24. Types of Genetic Marker
Single nucleotide polymorphism
A DNA sequence variation occurring when a
SINGLE nucleotide in the genome differs between
members of a biological species or paired
chromosomes in a human. IF two genes have a
SNP then they are referred to as alleles.
26. Types of Genetic Marker
Microsatellite repeat sequences
These are di-,tri- or tetra nucleotide tandem
repeats in DNA sequences. The number varies
within populations and within a persons alleles.
You can detect them by PCR as you will get
bigger and bigger products the more repeats you
have.
2
MRS
3
MRS
4
Fluorescent probe
30. Genetic Fingerprinting
Genetic fingerprinting is a powerful technique that
can allow identification of a person at a scene just
by comparing DNA of the person with some DNA
found at the scene
The first step is to RESTRICTION DIGEST (using
restriction endonucleases) chromosomal DNA, so
as to chop it into smaller pieces
Secondly, these fragments are separated
according to size using DNA GEL
ELECTROPHORESIS to produce a unique profile
31.
32. Probes and Fingerprinting
If you want to increase the reliability of the result
you can use DNA PROBES to further identify
specific bands within the gel
We can locate specific DNA fragments this way,
so if a particular RFLP is present, you can probe
for it
33. EXAMPLE
Which of these children is from the mother
previous marriage?
Which of these children is adopted?
