Polymerase chain reaction

2. A Brief History of PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College

3. Review: Th
e structure
of DNA
Double
Helix
Complementary Base Pairing

4. Review: Th
e structure
of DNA
Antiparallel Strands
Unzipping

5. The Proble
m
How do we identify a
nd detect a specific
sequence in a genom
e?

6. The Proble
m:
How do we identify and dete
ct a specific sequence in a
genome?
• TWO BIG ISSUES:
– There are a LOT of other sequences in a geno
me that we’re not interested in detecting. (SP
ECIFICITY)
– The amount of DNA in samples we’re interest
ed in is VERY small. (AMPLIFICATION)

7. The Proble
m:
Specificity
How do we identify and dete
ct a specific sequence in a
genome?
• Pine: 68 billion bp
• Corn: 5.0 billion bp
• Soybean: 1.1 billion bp
• Human: 3.4 billion bp
• Housefly: 900 million bp
• Rice: 400 million bp
• E. coli: 4.6 million bp
• HIV: 9.7 thousand bp

8. The Proble
m:
Specificity
• The human genome is 3.4 B bp
• If the bases were written in standard 10-point ty
pe, on a tape measure...
• ...The tape would stretch for 5,366 MILES!
• Identifying a 500bp sequence in a genome w
ould be like finding a section of this tape me
asure only 4 feet long!
Just How
Big Is 3.4
Billion?

9. The Proble
m:
Amplification
How many molec
ules do we need t
o be able to see t
hem?
• To be visible on an agarose gel, need around 10 ng
DNA for fluorescent stain (or around 25ng for Fast
Blast).
• For a 500-bp product band, weighing 660 g/mol.bp
, therefore need 10e-9 / (500*660) = 3.03e-14 mol
es.
• Avogadro’s number = 6.02e23.
• Therefore need 1.8e10 copies!
• In other words, to “see” a single “gene”, the DNA
in a sample of 100 cells would have to be multiplie
d 180 million times!!!!!

10. The Proble
m:
Specificity
Amplfication
• How do we identify and detect a specific s
equence in a genome?
• TWO BIG ISSUES:
– There are a LOT of other sequences in a geno
me that we’re not interested in detecting.
– The amount of DNA in samples we’re intereste
d in is VERY small.
PCR solves BOTH of thes
e issues!!!

11. So what’s P
CR used for
?
• Forensic DNA detection
• Identifying transgenic plants
• Detection and quantification of
viral infection
• Cloning
• Detection of ancient DNA
• Gene expression analysis

12. PCR History
The Inventi
on
In what has been called by some the greatest a
chievement of modern molecular biology, Kary
B. Mullis developed the polymerase chain re
action (PCR) in 1983. PCR allows the rapid synt
hesis of designated fragments of DNA. Using th
e technique, over one billion copies can be synt
hesized in a matter of hours.
PCR is valuable to scientists by assisting gene
mapping, the study of gene functions, cell iden
tification, and to forensic scientists in criminal id
entification. Cetus Corporation, Mullis' employer
at the time of his discovery, was the first to co
mmercialize the PCR process. In 1991, Cetus so
ld the PCR patent to Hoffman-La Roche for a pri
ce of $300 million. It is currently an indispensab
le tool for molecular biologists and the develop
ment of genetic engineering.
http://library.thinkquest.org/24355/data/details/1983a.html

13. Mr. PCR: K
ary B. Mulli
s
(1944 - )
The inventor of the DNA synthesis process known as the
Polymerase Chain Reaction (PCR). The process is an inva
luable tool to today's molecular biologists and biotechno
logy corporations.
Mullis, born in Lenoir, North Carolina, attended the Unive
rsity of Georgia Tech for his undergraduate work in che
mistry, and then obtained a Ph. D. in biochemistry from
Cal Berkeley.
In 1983, working for Cetus Corporation, Mullis develope
d the Polymerase Chain Reaction, a technique for the rap
id synthesis of a DNA sequence. The simple process invol
ved heating a vial containing the DNA fragment to split t
he two strands of the DNA molecule, adding oligonucleot
ide primers to bring about reproduction, and finally usin
g polymerase to replicate the DNA strands. Each cycle do
ubles the amount of DNA, so multiple cycles increase the
amount of DNA exponentially, creating huge numbers of
copies of the DNA fragment.
Mullis left Cetus in 1986. For his development of PCR, he
was co-awarded the Nobel Prize in chemistry in 1993.
http://library.thinkquest.org/24355/data/details/profiles/mullis.html

14. The Inventi
on of PCR
The process, which Dr. Mullis conceptualized in 1983, is h
ailed as one of the monumental scientific techniques of th
e twentieth century. A method of amplifying DNA, PCR mu
ltiplies a single, microscopic strand of the genetic materia
l billions of times within hours. Mullis explains:
http://www.osumu.org/mu/events_lectures1b.htm
"It was a chemical procedure that would make the structures of the
molecules of our genes as easy to see as billboards in the desert an
d as easy to manipulate as Tinkertoys....It would find infectious dise
ases by detecting the genes of pathogens that were difficult or impo
ssible to culture....The field of molecular paleobiology would blosso
m because of P.C.R. Its practitioners would inquire into the specific
s of evolution from the DNA in ancient specimens....And when DNA
was finally found on other planets, it would be P.C.R. that would tell
us whether we had been there before."

15. Mr. PCR: K
ary B. Mulli
s "Take all the MVPs from professional baseball,
basketball and football. Throw in a dozen favor
ite movie stars and a half-dozen rock stars for
good measure, add all the television anchor pe
ople now on the air and collectively we have n
ot affected the current good or the future welfa
re of mankind as much as Kary Mullis." -- Ted
Koppel, on ABC's "Nightline"
archive.salon.com/health/feature/2000/03/29/mullis/index.html

16. Practical Uses of PCR

17. Uses of PC
R:
Forensics
PCR’s ability to amplify even the smallest
amount of DNA from samples collected at
a crime scene gives the method great pow
er when used in criminal forensics.
The DNA from body fluid, hair, or other tis
sue samples is amplified to create a nearl
y unique pattern for each individual. This
pattern can then be compared to suspects
in the case.
The infamous OJ Simpson case was the fi
rst one in which the technique of PCR bec
ame widely publicized.

18. Uses of PC
R:
GMO Food
Detection
Genetically-modified foods (GMO foods) a
re widely grown in the USA and other cou
ntries.
For various reasons, some countries requi
re exporters to indicate the percentage of
GMO content in grain and food shipments.
PCR can be used to accurately measure t
he exact quantity of genetically-modified f
ood in a shipment, by “looking” at the DNA
that makes up the food!

19. Uses of PC
R:
Paternity T
esting
PCR’s power at identifying individual gene
tic makeup has made it invaluable for use
in paternity testing.
By amplifying specific DNA fragments fro
m parents or close relatives, it is possible t
o reconstruct relatedness between individ
uals.
PCR can not only identify relationships be
tween people today, but can also be used
to identify historical family relationships!

20. Uses of PCR
:
Archaeology
PCR has been used for many scientific st
udes in the field of archaeology:
Reconstructing the Dead Sea Scrolls.
Identification of paint pigments in cave pai
ntings.
Determining relatedness between individu
als in ancient ossuaries.
Constructing dinosaurs from blood preser
ved in amber specimens. (!)

21. Uses of PCR
:
Disease
Diagnosis
PCR is now invaluable in modern disease
diagnosis.
PCR can identify disease-causing organis
ms much earlier than other methods, sinc
e it looks for the DNA of the organism itsel
f, not its proteins or its effect on our immu
ne system.
PCR has even been used to diagnose dis
eases of the past, by amplifying minute a
mounts of disease-related DNA in preserv
ed specimens.

22. Uses of PCR
:
Disease
Treatment
PCR can not only be used in disease diag
nosis, but also as an aid in the treatment o
f diseases.
For example, real-time PCR is used to dir
ectly monitor the amount of HIV virus in p
atients suffering from infection. By monito
ring the amount of virus present, the drug
therapy can be continually adjusted to ma
ximize virus suppression.

23. Uses of PCR:
Wildlife Cons
ervation
Because PCR can be used to identify not
only individuals, but also can differentiate
between species, it is often used in wildlif
e conservation research.
PCR can be used to monitor trade in prod
ucts made from endangered species.
PCR can be used to monitor ecosystems f
or the presence of certain species.
PCR can be used even to monitor and ide
ntify indvidual animals!

24. The Human Genome Project has identified ten
s of thousands of genes in the human genome.
A key questions is: what do these genes do?
Part of the answer comes from determining wh
en the genes are turned on and off, and what a
ffects the level of gene expression. Quantitativ
e PCR is a key component of determining the l
evels of gene expression, and is a critical tool i
n cancer research, disease studies, and devel
opmental biology.
DNA
RNA
Enzymes
GENEX Analysis
Biology
Uses of PC
R:
Basic Researc
h

25. How PCR Works

26. How PCR w
orks
• Bio-Rad Animation
• Cold Spring Harbor Animation
• Animated .GIF files

27. Review: Th
e structure
of DNA
Double
Helix
Complementary Base Pairing

28. How PCR w
orks
• Cold Spring Harbor Animation

29. How PCR w
orks
• Bio-Rad PCR Animation

30. How PCR w
orks
• Animated .GIF #1

31. How PCR w
orks
• Animated .GIF #2

32. The PCR Reaction Chemistry

33. PCR Reactio
n Componen
ts
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• DNA Polymerase

34. PCR Reacti
on:
Water
• Water
– The medium for all o
ther components.

35. PCR Reacti
on:
Buffer
• Water
• Buffer
– Stabilizes the DNA polym
erase, DNA, and nucleotid
es
– 500 mM KCl
– 100 mM Tris-HCl, pH 8.3
– Triton X-100 or Tween

36. PCR Reacti
on:
Template D
NA
• Water
• Buffer
• DNA template
– Contains region to be a
mplified
– Any DNA desired
– Purity not required
– Should be free of polym
erase inhibitors

37. PCR Reacti
on:
Primers
• Water
• Buffer
• DNA template
• Primers
– Specific for ends of amp
lified region
– Forward and Reverse
– Annealing temps should
be known
•Depends on primer le
ngth, GC content, etc.
– Length 15-30 nt
– Conc 0.1 – 1.0 uM (pMol/
ul)

38. PCR Reacti
on:
Nucleotides
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
– Added to the growing cha
in
– Activated NTP’s
– dATP, dGTP, dCTP, dTTP
– Stored at 10mM, pH 7.0
– Add to 20-200 uM in assay

39. PCR Reacti
on:
Magnesium
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
– Essential co-factor of DNA polymer
ase
– Too little: Enzyme won’t work.
– Stabilizes the DNA double-helix
– Too much: DNA extra stable, non-s
pecific priming, band smearing
– Used at 0.5 to 3.5 uM in the assay

40. PCR Reacti
on:
Polymerase
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• DNA Polymerase
– The enzyme that do
es the extension
– TAQ or similar
– Heat-stable
– Approx 1 U / rxn

41. PCR Reaction
Components
Review
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• DNA Polymerase
Summary:

42. Setting Up PCR Reactions

43. A Typical P
CR Reactio
n
Sterile Water 38.0 ul
10X PCR Buffer 5.0 ul
MgCl2 (50mM) 2.5 ul
dNTP’s (10mM each) 1.0 ul
PrimerFWD (25 pmol/ul) 1.0 ul
PrimerREV 1.0 ul
DNA Polymerase 0.5 ul
DNA Template 1.0 ul
Total Volume 50.0 ul

44. Mixing Commo
n Reagents Sa
ves Time
Component 1X 20X
Sterile Water 38.0 ul 760 ul
10X PCR Buffer 5.0 ul 100 ul
MgCl2 (50mM) 2.5 ul 50 ul
dNTP’s (10mM each) 1.0 ul 20 ul
PrimerFWD (25 pmol/ul) 1.0 ul 20 ul
PrimerREV 1.0 ul 20 ul
DNA Polymerase 0.5 ul 10 ul
DNA Template 1.0 ul --
Total Volume 50.0 ul 980 ul
Aliquot
49 ul
Add DNA
as last step

45. An Even Si
mpler Appro
ach:
Mastermix
MASTERMIX 19.6 ul
Sterile Water
10X PCR Buffer
MgCl2
dNTP’s
DNA Polymerase
Primers Fwd+Rev 0.4 ul
DNA Template 20.0 ul
Total Volume 40.0 ul
Sterile Water
10X PCR Buffer
MgCl2
dNTP’s
DNA Polymerase
Primer FWD
Primer REV
DNA Template

46. Programming the Thermal Cycler

47. Typical The
rmal Cycler
Conditions
1. Initial Denaturation 95 C 3 min
2. DNA Denaturation 95 C 1 min
3. Primer Annealing 65 C 1 min
4. Primer Extension 72 C 1 min
5. Go to step #2, repeat 39 more times
6. End

48. Analyzing the Amplified DNA

49. PCR
Visualizing
Results
•After thermal cycling, tubes are take
n out of the PCR machine.
•Contents of tubes are loaded onto an
agarose gel.
•DNA is separated by size using an el
ectric field.
•DNA is then stained.
•PCR products are visible as different
“bands”.

50. PCR
Visualizing
Results

51. PCR
Visualizing
Results
Gel running

52. PCR
Visualizing
Results
After the gel has run, it is stained t
o reveal the DNA bands:

53. PCR
Visualizing
Results
The final result of the traditional P
CR procedure is a gel with a series
of bands:
Bands can be compared against e
ach other, and to known size-stan
dards, to determine the presence
or absence of a specific amplificat
ion product.

54. Webinars • Enzyme Kinetics — A Biofuels Case Study
• Real-Time PCR — What You Need To Know
and Why You Should Teach It!
• Proteins — Where DNA Takes on Form and
Function
• From plants to sequence: a six week colleg
e biology lab course
• From singleplex to multiplex: making the m
ost out of your realtime experiments
explorer.bio-rad.comSupportWebinars