AS Biology
Module 1
Section 1.3
DNA Technology
Specification
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.
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.
INITIAL DENATURATION
95-110 oC
PRIMERS ANNEAL
EXTENSION
DENATURATION
50-65 oC
FINAL EXTENSION
COOLING
72 oC
95-110 oC
72 oC
4-10 oC
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 template
molecule
Exercise
1.
Draw the primer for the
second DNA template
2.
Draw the sequence of
DNA produced
following DNA synthesis
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
The need for thermostability
Polymerase must
withstand this
temperature
But polymerase
only begins to
work here
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
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

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compatibility
Genetic fingerprinting at crime scenes
Paternity testing
DNA sequencing
DNA cloning
Creating large volumes of DNA for other work
Genetic mapping
PAST PAPER QUESTIONS
DNA Probes
 A DNA probe is a SHORT length of DNA with KNOWN
nucleotide BASE SEQUENCE
 Either has a
can be used as a marker
or
labelled end and so
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
The uses of DNA probes
PAST PAPER QUESTIONS
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
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
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
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.
Single nucleotide polymorphisms
 These can be detected by PCR
One type of DNA
One type of DNA
One type of DNA
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
Fluorescent probe
3 MRS
4 MRS
5 MRS
6 MRS
Microsatellite repeat sequences
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
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
EXAMPLE
 Which of these children is from the mother previous
marriage?
 Which of these children is adopted?
PAST PAPER QUESTIONS