Transcript An Introduction to PCR

An Introduction to PCR
Dave Palmer, Byotix, Inc.
Byotix, Inc.
1260 S. 47th St
Richmond, CA
Review: The structure of DNA
Helix
Complementary Base Pairing
Review: The structure of DNA
Unzipping
Antiparallel Strands
The Problem...
How do we identify and detect a
specific sequence in a genome?
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 we’re not interested in
detecting. (SPECIFICITY)
The amount of DNA in samples we’re
interested in is VERY small.
(AMPLIFICATION)
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.txt
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 )8633 Km!
Identifying a 500bp sequence in a
genome would be like finding a
section of this tape measure only
4 feet long...
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 / (500*660)
= 3.03e-14 moles
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 multiplied 180
million times!!!!!
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 we’re not interested in detecting.
The amount of DNA in samples we’re interested
in is VERY small.
PCR solves BOTH of these issues!!!
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.
Some Uses of PCR
Forensic DNA detection
Identifying transgenic
plants
Detection of viral infection
Cloning
Detection of ancient DNA
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.
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
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
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/020207/020207n6.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/mullis-autobio.html
Mr. PCR: Kary B. Mullis
"Take all the MVPs from professional baseball,
basketball and football. Throw in a dozen favorite
movie stars and a half-dozen rock stars for good
measure, add all the television anchor people now on
the air and collectively we have not affected the
current good or the future welfare of mankind as
much as Kary Mullis." -- Ted Koppel, on ABC's
"Nightline"
http://www.buzzle.com/editorials/3-29-2000-6929.asp
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
Uses of PCR: Ecology
With PCR, scientists can glean genetic information
from the faintest traces of the shyest, rarest animalurine, feces, scent marks, infinitesimal bits of hair
or skin rubbed onto a tree as the elusive creature
passes by. In addition to information that aids
classification, individuals can be identified so as to
estimate population size in a particular locale, or to
determine the geographic range of a single animal,
or a group of them. The technique can be adapted to
similar studies of plants, for analysis of patterns of
seed dispersal and the relative reproductive success
of specific plants. Researchers have even used PCR
to study badly damaged specimens such as roadkill,
or the leavings of carnivores, where little-known
vertebrates have been identified among the prey.
http://www.faseb.org/opar/bloodsupply/pcr.html
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
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
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
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. One
example is Kirk Bloodsworth. The Maryland
waterman was wrongly imprisoned for almost nine
years for the rape and murder of a 9-year-old girl,
but was freed in 1993 with the aid 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, as it did in Bloodsworth's case.
http://www.faseb.org/opar/bloodsupply/pcr.html
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
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 paleoartists 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
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 cancerridden 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
Today’s PCR: Population Genetics
Alu Insertion Polymorphism
detects the presence or absence of a
"jumping gene" on chromosome 16.
This simple genetic system has only two
alleles and three genotypes. Despite this
simplicity, allele frequencies vary
greatly in different world populations.
Alternate explanations about the causes
of this variation are consistent with
opposing theories of the origins of
modern humans
http://www.geneticorigins.org/geneticorigins/
Today’s PCR: Population Genetics
Fossil evidence:
5 million years ago: human lineage diverged from primates
1.5 million years ago: early hominids migrated out of
Africa to found populations in Europe, the Middle
East, and Asia
Conflicting Theories:
1) Did these early groups develop independently, and in
parallel, to become modern humans (multiregional
theory)?
2) Did a common ancestor develop in Africa, then migrate
worldwide and displace the early hominids
(displacement theory)?
http://www.geneticorigins.org/geneticorigins/
Today’s PCR: Population Genetics
Alu is an example of a so-called "jumping gene" – a transposable DNA
sequence that "reproduces" by copying itself and inserting into new
chromosome locations.
Alu elements are classified as SINEs, or Short INterspersed Elements. All
Alus are approximately 300 bp in length and derive their name from a single
recognition site for the restriction enzyme AluI located near the middle of the
Alu element. Human chromosomes contain about 1,000,000 Alu copies,
which equal 10% of the total genome.
Once an Alu integrates into a new site, it accumulates new mutations at the same rate as surrounding DNA loci.
Alu elements can be sorted into distinct lineages, or families, according to inherited patterns of new mutations.
These studies suggest that the rate of Alu transposition has changed over time – from about one new jump in
every live birth, early in primate evolution, to about one in every 200 newborns today.
http://www.geneticorigins.org/geneticorigins/
Today’s PCR: Population Genetics
Each Alu is the "fossil" of a unique transposition event that
occurred only once in primate evolution. Thus, all primates
showing an Alu insertion at a particular locus have
inherited it from a common ancestor. This is called identity
by descent.
Most Alu mutations are "fixed," meaning that both of the
paired chromosomes have an insertion at the same locus
(position). However, a number of human-specific Alus are
dimorphic – an insertion may be present or absent on each
of the paired chromosomes of different people. These
dimorphic Alus inserted within the last million years,
during the evolution and dispersion of modern humans.
These dimorphisms show differences in allele and genotype
frequencies between modern populations and are tools for
reconstructing human prehistory.
http://www.geneticorigins.org/geneticorigins/
Today’s PCR: Population Genetics
Today, we’ll isolate our own DNA from cheek cells,
Amplify the PV92 region of our DNA with PCR,
And determine (using a gel) if we have an Alu insertion
there.
+/+
or
+/-
or
-/-
The ratios of these genotypes, over a whole population, can
help determine the relatedness to other populations.
http://www.geneticorigins.org/geneticorigins/