Genetics Techniques:

RFLP & PCR

AP Biology
Unit 3

RFLP
Stands for Restriction How many fragments will result
when each of these alleles are
Fragment Length
digested with DdeI?
Polymorphism
3 fragments
Takes advantage of
differences in DNA
between individuals that
result in different
fragments when digested
with restriction enzymes
2 fragments

RFLP
To see RFLP, DNA is
digested with the
appropriate restriction
enzymes and run on an
agarose gel.
A Southern Blot is
performed to complete
the analysis.

Southern Blotting
A method to visualize
specific segments of DNA
usually a particular gene.
Uses radioactive probes
that bind to the specific
DNA segments
Ex. When testing for the
hemoglobin alleles, the
probe would bind to these
regions

Southern Blotting
Steps:
Soak gel in basic solution to separate DNA
strands
Transfer DNA on to a nylon membrane
(spacing of DNA is maintained)
Incubate with radioactive probe for specific
segment
Wash away unbound probe
Detect probes using x-ray film
autoradiograph

RFLP Animations
Animation #1
Animation #2

Polymerase Chain Reaction

PCR allows scientists to amplify small,
specific segments of DNA = make millions
of copies of segment
Allows for amplification at an exponential
rate
DNA Replication in a test tube

Materials needed for PCR

Target DNA (the DNA you want to copy)

Free Nucleotides (A, T, C, G)
Primers (DNA primers, not RNA)
Taq Polymerase (heat stable DNA Polymerase
III)
Mg2+ (cofactor that DNA Polymerase III needs
to work)
Buffer
Thermocycler (machine that changes
temperatures)

Overview of PCR
Uses different temperatures to amplify
DNA
Step 1: Separate existing DNA strands
95C (Denaturation)
Step 2: Lower temperature to allow primers
to bind to target DNA 55C (Annealing)
Step 3: Raise temperature to allow Taq
Polymerase to build DNA strand 72C
(Extension)

Differences between DNA

Replication & PCR
No Helicase or Topoisomerase PCR uses
the first heat step to completely separate the
strands of DNA
No Primase primers are already made
DNA primers (not RNA) no need for
DNA Polymerase I
No leading or lagging strands DNA is
completely unzipped, no Okazaki fragments

PCR animation
animation