An Introduction to PCR

1
An Introduction to PCR

• Dave Palmer
• Technical Support
• Bio-Rad Laboratories

2
Dave Palmer

• Plant Protection (U of Guelph)
• Molecular Plant Pathology (Oklahoma State)
• Field Research Biologist (Zeneca)
• Transgenic Research Scientist (Syngenta)
• Ag-Biotech Company Founder (Byotix)
• Nucleic Acid Applications (Bio-Rad)

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Review The structure of DNA
Helix
Complementary Base Pairing
4
Review The structure of DNA
Unzipping
Antiparallel Strands
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The Problem...

• How do we identify and detect a specific sequence
  in a genome?

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The Problem...
(How do we identify and detect a specific
sequence in a genome?)

• TWO BIG ISSUES
• There are a LOT of other sequences in a genome
  that were not interested in detecting.
  (SPECIFICITY)
• The amount of DNA in samples were interested in
  is VERY small. (AMPLIFICATION)

7
Review Genome Sizes

• 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

http//www.cbs.dtu.dk/databases/DOGS/abbr_table.tx
t
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Just How Big Is 3.4 Billion?

• Human genome is 3.4 B bp
• If the bases were written in standard 10-point
  type, on a tape measure...
• ...The tape would stretch for 5,366 MILES!
• Identifying a 500bp sequence in a genome would be
  like finding a section of this tape measure only
  4 feet long!

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How many molecules do we need to be able to see
them?

• To be visible on an agarose gel, need around 10
  ng DNA
• For a 500-bp product band, weighing 660 g/mol.bp,
  therefore need 10e-9 / (500660) 3.03e-14 moles
• Avogadros 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
  multiplied 180 million times!!!!!

10
The Problem...

• How do we identify and detect a specific sequence
  in a genome?
• TWO BIG ISSUES
• There are a LOT of other sequences in a genome
  that were not interested in detecting.
• The amount of DNA in samples were interested in
  is VERY small.
• PCR solves BOTH of these issues!!!

SPECIFICITY
AMPLIFICATION
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PCR History
In what has been called by some the greatest
achievement of modern molecular biology, Kary B.
Mullis developed the polymerase chain reaction
(PCR) in 1983. PCR allows the rapid synthesis of
designated fragments of DNA. Using the technique,
over one billion copies can be synthesized in a
matter of hours. PCR is valuable to scientists
by assisting gene mapping, the study of gene
functions, cell identification, and to forensic
scientists in criminal identification. Cetus
Corporation, Mullis' employer at the time of his
discovery, was the first to commercialize the PCR
process. In 1991, Cetus sold the PCR patent to
Hoffman-La Roche for a price of 300 million. It
is currently an indispensable tool for molecular
biologists and the development of genetic
engineering.  
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Some Uses of PCR

• Forensic DNA detection
• Identifying transgenic plants
• Detection and quantification of viral infection
• Cloning
• Detection of ancient DNA
• Gene expression analysis

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Mr. PCR Kary B. Mullis
(1944 - ) The inventor of the DNA synthesis
process known as the Polymerase Chain Reaction
(PCR). The process is an invaluable tool to
today's molecular biologists and biotechnology
corporations.                  Mullis, born in
Lenoir, North Carolina, attended the University
of Georgia Tech for his undergraduate work in
chemistry, and then obtained a Ph. D. in
biochemistry from Cal Berkeley.
                           In 1983, working for
Cetus Corporation, Mullis developed the
Polymerase Chain Reaction, a technique for the
rapid synthesis of a DNA sequence. The simple
process involved heating a vial containing the
DNA fragment to split the two strands of the DNA
molecule, adding oligonucleotide primers to bring
about reproduction, and finally using polymerase
to replicate the DNA strands. Each cycle doubles
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. Mullis is currently doing HIV and AIDS
research.  
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The Invention of PCR
The process, which Dr. Mullis conceptualized in
1983, is hailed as one of the monumental
scientific techniques of the twentieth century. A
method of amplifying DNA, PCR multiplies a
single, microscopic strand of the genetic
material billions of times within hours. Mullis
explains
 
"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 and as easy to
manipulate as Tinkertoys....It would find
infectious diseases by detecting the genes of
pathogens that were difficult or impossible to
culture....The field of molecular paleobiology
would blossom because of P.C.R. Its practitioners
would inquire into the specifics 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."
http//www.osumu.org/mu/events_lectures1b.htm
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The Invention of PCR
 
Mullis's little silver Honda Civic was purring
through the vineyards and redwoods of the
Anderson Valley and his mind wandered. Life is
sweet, he thought 'I am a big kid with a new car
and a full tank of gas. I have shoes that fit. I
have a woman sleeping next to me and an exciting
problem, a big one.' At mile-marker 46.58 on
Highway 128 - he had both the presence of mind
and the sense of history to note the exact spot,
if not the month - the epiphany arrives. 'Holy
s__,' Mullis cries out, and his girlfriend
almost, but not quite, wakes up. He pulls the
Honda to the side of the road to write down his
ideas and check his calculations. Within feverish
minutes, the problem is solved, and Mullis is
left with the mop-up operation of getting PCR
actually to work. This takes almost two years,
and the original report was famously rejected by
both Nature and Science. Mullis was not fazed
'"F___ them," I said.
http//www.lrb.co.uk/v21/n13/shap2113.htm
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Kary Mullis Trivia

• Mullis repeatedly asserts that he was ripped off
  financially by the greedy confederacy of dunces
  who were his colleagues at Cetus. He got a
  10,000 bonus, and Cetus cleared 300 million
  when the patent rights to PCR were sold to
  Hoffman-LaRoche - possibly the most ever paid for
  a patent. (http//barometer.orst.edu/0102/02winter
  /020207/020207n6.html)
• A few years ago he started up a company -
  GeneStones - that would copy the DNA in hair or
  skin samples of famous people, multiply it by
  PCR, and then implant it in artificial gemstones,
  where it would appear as 'a white, ethereal
  cloud'. Depending on the setting and
  production-run, 75 to 200 would get you John F.
  Kennedy, Napoleon or Marilyn Monroe on your
  finger. (http//barometer.orst.edu/0102/02winter/0
  20207/020207n6.html)
• His first published scientific paper, in the
  premier scientific journal Nature in 1986,
  described how he viewed the universe while on LSD
  - pocked with black holes containing antimatter,
  for which time runs backward. He has been known
  to show photographs of nude girlfriends during
  his lectures. (http//www.virusmyth.net/aids/data/
  cfmullis.htm)
• We tortured the cows. We sliced apples and
  slipped them onto the electric fence that
  contained them in the newer parts of the pasture.
  Cows like apples and they kept trying. K.
  Mullis,www.nobel.se/chemistry/laureates/1993/mulli
  s-autobio.html

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Mr. PCR Kary B. Mullis
 
http//www.buzzle.com/editorials/3-29-2000-6929.as
p
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Uses of PCR Ancient DNA
Archaeologists have happily seized on PCR and are
applying it in an amazing variety of ways. It is
helping, for example, to launch a new chapter in
the colorful and controversial story of the
2000-year-old Dead Sea Scrolls, which are written
on parchment made out of skins from goats and
gazelles. Researchers are analyzing the parchment
fragments to try to identify individual animals
they came from. The hope is that the genetic
information will guide them in piecing together
the 10,000 particles of scrolls that remain.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Disease Detection
PCR can also be more accurate than standard
tests. It is making a difference, for example, in
a painful, serious, and often stubborn misfortune
of childhood, the middle ear infection known as
otitis media. The technique has detected
bacterial DNA in children's middle ear fluid,
signaling an active infection even when culture
methods failed to detect it. Lyme disease, the
painful joint inflammation caused by bacteria
transmitted through tick bites, is usually
diagnosed on the basis of symptom patterns. But
PCR can zero in on the disease organism's DNA
contained in joint fluid, permitting speedy
treatment that can prevent serious complications.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Endangered Species
Researchers have used the technique to aid in
reducing illegal trade in endangered species, and
products made from them. Because PCR is a
relatively low-cost and portable technology, and
likely to become more so, it is adaptable for
field studies of all kinds in the developing
countries. It is also a tool for monitoring the
release of genetically engineered organisms into
the environment.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Forensic DNA
The technique's unparallelled ability to identify
and copy the tiniest amounts of even old and
damaged DNA has proved exceptionally valuable in
the law, especially the criminal law. PCR is an
indispensable adjunct to forensic DNA
typing-commonly called DNA fingerprinting.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Proving Innocence
DNA typing is only one of many pieces of evidence
that can lead to a conviction, but it has proved
invaluable in demonstrating innocence. Dozens of
such cases have involved people who have spent
years in jail for crimes they did not commit.
Many people have been freed because of the power
of PCR. Even when evidence such as semen and
blood stains is years old, PCR can make unlimited
copies of the tiny amounts of DNA remaining in
the stains for typing.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Disease Detection
The method is especially useful for searching out
disease organisms that are difficult or
impossible to culture, such as many kinds of
bacteria, fungi, and viruses, because it can
generate analyzable quantities of the organism's
genetic material for identification. It can, for
example, detect the AIDS virus sooner during the
first few weeks after infection than the standard
ELISA test. PCR looks directly for the virus's
unique DNA, instead of the method employed by the
standard test, which looks for indirect evidence
that the virus is present by searching for
antibodies the body has made against it.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Ancient DNA
Archaeologists are finding that PCR can
illuminate human cultural practices as well as
human biology. Analyzing pigments from
4000-year-old rock paintings in Texas, they found
one of the components to be DNA, probably from
bison. The animals did not live near the Pecos
River at that time, so the paleo-artists must
have gone to some effort to obtain such an
unusual ingredient for their paint. Taking so
much trouble suggests that the paintings were not
simply decorations, but had religious or magical
significance.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Disease Detection
PCR can even diagnose the diseases of the past.
Former vice president and presidential candidate
Hubert H. Humphrey underwent tests for bladder
cancer in 1967. Although the tests were negative,
he died of the disease in 1978. In 1994,
researchers compared a 1976 tissue sample from
his cancer-ridden bladder with his 1967 urine
sample. With the help of PCR amplification of the
small amount of DNA in the 27-year-old urine,
they found identical mutations in the p53 gene,
well-known for suppressing tumors, in both
samples. "Humphrey's examination in 1967 may have
revealed the cancerous growth if the techniques
of molecular biology were as well understood then
as they have become," the researchers said.
http//www.faseb.org/opar/bloodsupply/pcr.html
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Uses of PCR Gene Expression Analysis
The Human Genome Project has identified tens of
thousands of genes in the human genome. A key
questions is what do these genes do? Part of
the answer comes from determining when the genes
are turned on and off, and what affects the level
of gene expression. Quantitative PCR is a key
component of determining the levels of gene
expression, and is a critical tool in cancer
research, disease studies, and developmental
biology.
DNA
Enzymes
RNA
GENEX Analysis
Biology
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Todays PCR GMO Detection

• What are GM crops?
• Detecting GM crops
• Elisa, PCR
• PCR Analysis
• Sequences detected (PSII, 35S, NOS)
• Controls (non-GM, GM-positive)
• Reaction sets