1. Advanced Molecular Biological Techniques

2. 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?

3. 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

4. PCR Denaturation Temperature is increased to separate the DNA strands
HEAT separates the strands
Denaturation
Temperature is increased to separate the DNA strands
94-96 degrees breaks hydrogen bonds between the nitrogenous base pairs

5. PCR Annealing 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

6. 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'

7. 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'
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’A A G T G 5'
A A G T G 5'
5' G C T T G 3’
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' …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'

8. PCR
Extension
Taq polymerase builds complimentary strands using free nucleotids
Extracted from Thermus aquaticus

9. DNA is heated to 95oC again and denatured.
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 G C T T G C A A T C C G A T T C A C C G C T …3'
C A A T C C G A T T C A C 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' …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'
3’ C G A A C G T T A G G C T
C A A T C C G A T T C A C C G C T… 3’
Notice the appearance of strands of DNA of different length. These are referred to as “variable length fragments.
5' G C T T G
A A G T G 5'
5' G C T T G
A A G T G 5'

10. PCR Exponential Amplification
Process is repeated and region of interest is amplified exponentially.
Minutes per cycle

11. “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

12. 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.

13. 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.

14. “Restriction Fragment Length Polymorphism” (RFLP)
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

15. “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

16. “Restriction Fragment Length Polymorphism” (RFLP)
DNA digested with restriction endonuclease(s)
Cut DNA at specific palindromic points
Left with sticky ends

17. “Restriction Fragment Length Polymorphism” (RFLP)
Gel Electrophoresis
Fragments separate based on size and charge

18. “Restriction Fragment Length Polymorphism” (RFLP)
DNA denatured into single strands
Southern blotting.
Banding patterns transferred from gel to nylon membrane using electric current

19. “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)

20. “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.

21. RFLP Analysis
Southern Blot

22. Homework

23. APPLICATIONS
Gene therapy
PCR applications
RFLP applications

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. Forensic investigations and Paternity testing
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

31. 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?

32. 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

33. Who da babydaddy??? IS THE ALLEGED FATHER THE BABYDADDY??
Assign “names” to RFLP variants
Determine genotypes of sources
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
Source
Genotype
Mother
B/E
Child
B/D
Alleged father (A.F.)
A/C
Child/A.F. mix
A/B/C/D A B C D E
IS THE ALLEGED FATHER THE BABYDADDY??
 NO! Follow link for more detail

34. IS THE ALLEGED FATHER THE BABYDADDY??  YES!
Source
Genotype
Mother
B/D
Child
C/D
Alleged father (A.F.)
A/C
Child/A.F. mix
A/C/D A B C D
IS THE ALLEGED FATHER THE BABYDADDY??
 YES!

35. 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?

36. PCR Animations http://www.dnalc.org/ddnalc/resources/animations.html