PCR (polymerase chain reaction) was invented by Kary Mullis in 1987 and allows for the specific amplification of a single DNA sequence. It takes advantage of basic DNA replication requirements by using DNA polymerase, primers, nucleotides, and thermal cycling to exponentially amplify the target sequence. Key aspects of PCR include using specific primers, thermocycling between denaturation, annealing, and extension steps, and the use of Taq polymerase which functions at high temperatures. PCR has many applications including cloning, detection of genetic diseases, forensics, and quantification of DNA or RNA.

1. PCR
Polymerase Chain Reaction

2. Invented by Kary Mullis
Mullis and Faloona, 1987. Specific
synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction.
Nobel Prize 1993

3. Kary Mullis himself….

4. “I was working for Cetus, making oligonucleotides.
They were heady times. Biotechnology was in flower
and one spring night while the California buckeyes
were also in flower I came across the polymerase
chain reaction. I was driving with Jennifer Barnett to
a cabin I had been building in northern California.
She and I had worked and lived together for two
years. She was an inspiration to me during that time
as only a woman with brains, in the bloom of her
womanhood, can be. That morning she had no idea
what had just happened. I had an inkling. It was the
first day of the rest of my life.”
- from Karry Mullis’s autobiography at the Nobel e-Museum

5. PCR
Specifically targets and amplifies a
SINGLE sequence from within a complex
mixture of DNA.
How is this different from cloning?

6. Takes advantage of basic
requirements of replication
A DNA template
Nucleotides
Primers
polymerase
PCR is DNA replication in a test tube

7. Primers
Must have some information about
sequence flanking your target
Primers provide specificity

8. 5’ 3’
3’ 5’
Complementary to opposite strands with 3’
ends pointing towards each other
Should have similar melting temperatures
Be in vast excess

9. Melting temperature
TmoC = 2(A/T) + 4(G/C)
TmoC Temperature at which
half possible H bonds are
formed

10. The basic process
dsDNA
Denature (95 degrees)

11. 5’ 3’
3’ 5’
http://www.dnalc.org/shockwave/pcranwhole.html

12. Thermocycling
94 degrees
55 degrees
70 degree

13. Heat-stable polymerase is vital
to the ease of the process…

14. Thermus aquaticus:

15. The Thermus
aquaticus DNA
polymerase
Taq
Not permanently
destroyed at 94ºC
Optimal
temperature is 72ºC

16. Problems with Taq
Does not have proof readng ability
Error rate 1 in 2 X 104 bases
Seems rare but can be recovered in
cloning a single molecule
Newer polymerases have high fidelity

17. Termplates for PCR
Small amount of template
In theory a single molecule
Do not need to isolate sequence of
interest
DNA template need not be highly
purified
DNA is stable in absence of nucleases

18. Templates for PCR
Dried blood
Semen stains

20. Templates for PCR
Dried blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes

21. PCR variations
Add 5’ extensions for cloning
3’
5’
3’ 5’
5’
G
A
A
T
T 3’
C
3’ C
T
T
A
A
G 5’

22. Cloning PCR Fragments
Taq leaves 3’ A overhang.
A
A
T
T

23. rtPCR
Reverse trancriptase PCR
Use mRNA as a template
Isolated cDNA clones
Can be quantitative

25. Inverse PCR
unknown
known
unknown
Known
unknown

26. known
unknown

27. Nested primers
PCR primers are not always an exact match!
Degeneracy
Lower annealing temperatures increase chances of
amplifying something!
Could be wrong thing!

28. Nested primers
2
1
2 1

29. Quantitative PCR

30. Real Time PCR
Detection and quantitation of fluorescent reporter the
signal of which increases in direct proportion to the
amount of PCR product in a reaction
Does not measure the amount of end product but its
production in real time

31. SYBR
green
Also binds
primer dimers
Can
overestimate
product

32. Molecular Beacons
Uses FRET
Fuorescence Resonance Energy Transfer
Uses two sequence specific
oligonucleotides labeled with fluorescent
dyes

34. Taq Man

35. Other application of PCR
Detection of mutations
screen for inherited disorders
Detection of HIV
Not standard test given
Detect tuberculosis without culturing
Prenatal sex determination
DZY1 = Y specific sequence present in
5000 copies

36. Other application of PCR
Preimplantation diagnosis of genetic
diseases
Forensics
Paternity testing

37. Forensics
STR
Short Tandem Repeats
2 to 7 base pairs repeated 7-40 times
Replaced VNTRs in forensic analysis
13 Highly polymorphic loci have been
selected by FBI
Population match probabilities 0.1 - 0.28
Probability One in 5.7 X 10-15
Combined DNA Index System (CODIS)

39. STR analysis of family of last
Tsar of Russia

40. VNTR’s
Can use PCR to visualize VNTRs
Eg. pMCT118 in chromosome 1

41. VNTR analysis

42. Problems with PCR
Contamination
Theoretically one molecule can amplify
Takes one mismatch early on to amplify
the wrong fragment