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Advanced Molecular
Biological Techniques
C.S.I
• How do crime scene
investigators (like
Dexter) perform so
many genetics tests
when they often only
find one cell at the
scene?
• How do C.S.I’s identify
suspects through
DNA?
Polymerase Chain
Reaction (PCR)
– Mullis first proposed PCR in
1987
• Allows creation of more copies
of DNA (20 cycles can give
over a million copies!)
• Similar to DNA Replication
• PCR Animation
PCR
HEAT
separates the
strands
• Denaturation
• Temperature is increased to separate the DNA
strands
– 94-96 degrees breaks hydrogen bonds between
the nitrogenous base pairs
• Annealing
PCR
– DNA Primers anneal
• Temperature drops to 50-65 degrees
• Added to 3’ end of specific sequence (region of
interest)
• Both strands of DNA are used.
5’-3’ DNA primers
Suppose this is the portion of DNA to be amplified.
Cycle 1: The DNA is denatured by heating to 95oC
DNA primers are included with the DNA sample.
DNA primers bind to DNA when cooled to 60oC
Taq polymerase along with deoxynucleotides are also part of the mixture.
5' …A T G C T T G C A A T C C G A T T C A C C G C T …3'
5' G C T T G 3'
3' A A G T G 5'
3' …T A C G A A C G T T A G G C T A A G T G G C G A… 5'
Cycle 1:
When the mixture is warmed to 72oC, Taq polymerase produces the complementary DNA strand in
the 5’ to 3’ direction.
5' …A T G C T T G C A A T C C G A T T C A C C G C T …3'
3’…T A C G A A C G T T A G G C T
3’AAAAGGTTGG5'5'
5' G C T T G 3’
C A A T C C G A T T C A C C G C T…3’
3' …T A C G A A C G T T A G G C T A A G T G G C G A… 5'
PCR
• Extension
– Taq polymerase builds complimentary strands
using free nucleotids
• Extracted from Thermus aquaticus
Cycle 2:
DNA is heated to 95oC again and denatured.
Primers are annealled to DNA strands when cooled to 60oC.
Taq polymerase extends the strands when warmed to 72oC.
3’ … T A C G A A C G T T A G G C T
5' …A T GC CATATTGC CCAGAATTCTCCGA AC T3’T C A C C G C T …3'
3’…T A C G A A C G T T A G G C T
A A G T G 5'
5' G C T T G
C A A T C C G A T T C A C C G C T…3’
3’ C G A A C G T3'T …T
A GAGCCGTA A C G T T A G G C T A A G T G G C G A… 5'
C A A T C C G A T T C A C C G C T… 3’
G C T T G of strands of DNA of different length. These are referred to as “variable
A A G T length
G 5' fragments.
Notice the 5'
appearance
5' G C T T G
A A G T G 5'
PCR
• Exponential Amplification
– Process is repeated and region of interest is
amplified exponentially.
– Minutes per cycle
“Restriction Fragment Length
Polymorphism”
(RFLP)
– the difference in DNA fragment lengths (when cut
by restriction enzymes) between individuals
• Allows scientists to match DNA from a crime
scene to a suspect, obtain diagnosis of genetic
diseases, and to determine
paternity/maternity
RFLP animation
The Thermal Cycler
• The repetitive nature of heating
and cooling lends itself to
automation.
• Table top thermal cyclers are no
larger than a bread-baking
machine.
• The thermal cycler will take a
mixture consisting of the original
DNA sample, Taq polymerase,
forward and reverse primers and
free deoxynucleotides, all in a 0.5
mL tube and produce a billion
copies in just a few hours.
The Thermal Cycler
• Note the display:
–
–
–
–
the thermal cycler is programmed for 20 cycles
DNA is denatured at 94°C for 30 s
primers anneal at 55°C for 1 minute
and Taq polymerase is given 3 minutes at 68°C to extend
the primers and produce the complementary strand.
“Restriction Fragment Length
Polymorphism”
(RFLP)
• Polymorphism
– Differences in a DNA sequence
between individuals
– Organisms of the same species
carry the same genes but differ
in their respective alleles.
– Genomes of individuals of the
same species are polymorphic
(unless identical twins)
POLYMORPHISM - any detectable difference in DNA
“Restriction Fragment Length
Polymorphism”
(RFLP)
• Polymorphism in exons.
– To identify individuals with specific mutations.
• Possible polymorphism in introns (VNTRs)
– Variable number tandem repeats
• TAGTAGTAGTAGTAG….
– Unique to individuals
“Restriction Fragment Length
Polymorphism”
(RFLP)
• DNA digested with restriction endonuclease(s)
– Cut DNA at specific palindromic points
– Left with sticky ends
“Restriction Fragment Length
Polymorphism”
(RFLP)
• Gel Electrophoresis
– Fragments separate based on size and charge
“Restriction Fragment Length
Polymorphism”
(RFLP)
• DNA denatured into single strands
• Southern blotting.
– Banding patterns transferred from gel to nylon
membrane using electric current
“Restriction Fragment Length
Polymorphism”
(RFLP)
• Membrane soaked in a solution containing
radioactive complimentary nucleotide probes.
– DNA sequence tagged
– Base pairing occurs between target DNA and probe
(known as hybridization)
“Restriction Fragment Length
Polymorphism”
(RFLP)
• Autoradiogram produced
– Nylon membrane placed against X-ray film for 2-3
weeks.
– Probes burn image on to the film.
RFLP Analysis
Southern Blot
Homework
APPLICATIONS
•Gene therapy
•PCR applications
•RFLP applications
Gene therapy
• Refers to any method for treating genetic
diseases that involves altering the DNA
sequence
– Inserting genes
– Deleting genes
– Altering expression of genes
• Can act on either the germ line cells (results
will be heritable), or the somatic cells
Insertion
• Inserting genes can be accomplished by
introducing vectors into the host cell
– Viral transfection
– Direct injection of DNA
• Insertion can occur at a random location: risk
of altering existing host gene
Altering expression
• Use an antisense oligonucleotide
– “oligonucleotide” – A short nucleic acid (RNA) strand
– “antisense” – Complementary to a functional mRNA
• Introduce short antisense RNA strands
• Complementary base-pairing with mRNA will occur 
prevents translation
• Use to de-activate specific mRNA’s associated with disease
• Effectiveness of antisense gene therapy has
so far been limited
• Clinical trials:
– HIV/AIDS
– Cancer
– High cholesterol
– Ebola hemorrhagic fever
– Pain management in cancer patients
• Read section 6.4 to find out more about this
Applications of PCR
• Useful when only a small
amount of DNA is available
– Archaeological samples
• “degraded DNA"
– Forensic investigations
• DNA evidence may be limited
• Medical diagnosis
– e.g., HIV virus. Amplification allows detection
before immune system symptoms are
widespread
Applications of RFLP
Genetic screening
• Some genetic diseases are associated with particular RFLP
banding patterns
– e.g., Sickle cell anemia – base pair substitution occurs within
restriction site for DdeI
• Similar techniques can be used to screen for known genetic
mutations
– Digest DNA and hybridize probes that are complementary to
mutations
– Requires blood sample or another biological sample
– Prenatal screening: use amniotic fluid
DNA Fingerprinting
• Forensic investigations and Paternity testing
• Location of restriction sites is unique to
individuals
• Digest genomic DNA with several RE’s
– Banding pattern should be particular to each
individual
• Compare suspect banding patterns with those
from crime scene samples or from child
– Forensics: Looking for 100% concordance
– Paternity: Looking for 50% concordance
Side note: DNA profiles today...
• RFLP is time-consuming and requires large
amounts of DNA
• PCR-based techniques are actually used today
for generating DNA profiles
Why do you think RFLP-based
DNA fingerprinting is an
unattractive alternative for
forensic investigations?
• VNTR’s (microsatellites) are the markers of
choice
– The copy number will vary between individuals
• PCR is used to selectively amplify certain
VNTR loci so the number of repeats can be
determined
• Separation occurs by electrophoresis, but
within a narrow glass capillary tube instead
of a slab of gel
Who da babydaddy???
 Assign “names” to RFLP variants
 Determine genotypes of sources
A
B
C
D
Source
Genotype
Mother
B/E
Child
B/D
Alleged father
(A.F.)
A/C
Child/A.F. mix
A/B/C/D
 Compare: Child should share one RFLP variant
with father, one with mother
 As a rule, Child/AF mix should not have more
than three bands
E
IS THE ALLEGED FATHER THE BABYDADDY??
 NO!
Follow link for more detail
A
B
C
D
Source
Genotype
Mother
B/D
Child
C/D
Alleged father
(A.F.)
A/C
Child/A.F. mix
A/C/D
IS THE ALLEGED FATHER THE
BABYDADDY??
 YES!
To catch a killer...
• Two suspects
• Two samples recovered from scene
• Victim shares no bands with either
suspect
• Crime Scene 2 sample:
– Victim is the source
• Crime Scene 1 sample:
– Whodunnit?
PCR Animations
• http://www.dnalc.org/ddnalc/resources/animations.html
• http://www.amnh.org/learn/pd/genetics/pcr/interactive.html