Polymerase
Chain Reaction (PCR)
Objectives
Discussion
Questions
Glossary
Interesting web sites
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During this lecture you
will learn the steps of PCR and how huge quantities of a particular section
of DNA can be produced from a very small initial amount of genetic material
Objectives:
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· Learn the terms
primer, annealing, denaturing, elongation, and Taq polymerase. Understand
how Taq polymerase adds nucleotides to single-stranded DNA.
· Learn how time and temperature control specificity, so the right
DNA sequence is amplified.
· Understand the powerful, exponential nature of the process.
Discussion:
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At times scientists need
many copies of a particular segment of DNA in order to perform a test or experiment.
They may need to determine whether a person has a genetic mutation that causes
a disease, or whether a person is infected with a particular bacteria or virus.
As you may have seen in the news, they may want to use a tiny bit of blood or
other evidence to help police determine whether to charge someone with a crime.
To get the copies they need, scientists can use polymerase chain reaction -
PCR - to create more copies of the segment of DNA in which they're interested.
Finding a gene or other
specific sequence of DNA can be compared to finding the proverbial needle in
the haystack. But what if you could manipulate the haystack so that it produced
millions of copies of the needle? That's what PCR does, using time, temperature
and some of the same molecular tools that Nature created to replicate DNA.
Very little DNA containing
the sequence to be amplified is needed to begin PCR - in fact, PCR can be accomplished
with just one DNA molecule. Lots of bases (A, C, G, and T), oligonucleotide
primers - singled-stranded sections of DNA - and DNA polymerase are added to
the tube containing the DNA.
When the mixture is heated
to about 94 degrees C for 5 minutes, the two strands of the DNA helix separate.
This is called "denaturing." The strands serve as the first templates
for the DNA synthesis that occurs later in the process. For the next step in
the process to occur, the solution is cooled to about 65 degrees C. At this
temperature, the oligonucleotide primers attach themselves - "anneal"
- to the proper locations on the strands of DNA. Because the DNA bases always
pair with their complementary bases (A with T and G with C) the primers can
be constructed to connect to specific locations on the DNA strands.
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Now it's time for
the DNA polymerase to go to work, once the temperature is raised to 72
degrees C. Like a bricklayer matching a pattern, the DNA polymerase goes
to work at the 3' end of the oligonucleotide primer, attaching the proper
bases to their complementary bases on the single strand of DNA, moving
in the 5' to 3' direction. Eventually, the two single strands of DNA become
two double strands of DNA. Then the mixture is re-heated to 94 degrees
C, and the process repeats.
Early on, DNA polymerase
from E. coli bacteria was used in PCR. However, each time the cycle returned
to the 94 degrees C step necessary to denature the DNA, the E. coli DNA
polymerase was destroyed. That meant new DNA polymerase had to be added
at each cycle. That tedious process was eliminated with the discovery
of Taq polymerase, taken from bacteria that live in hot springs. Taq polymerase
can survive the high temperatures necessary for DNA denaturing, which
has allowed the PCR process to be automated.
Each cycle, the number
of double-stranded DNA fragments doubles. This is a powerful amplification
process: After just 32 cycles one fragment has been transformed into more
than 1 million fragments - hopefully identical fragments. This amplification
power does have implications for mistakes or contamination that might
occur during the process.
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Questions:
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1. At what temperature does
DNA denature (separate into single strands)?
2. Where does Taq polymerase come from and what makes it well suited to PCR?
3. Why is it important to avoid contamination or other PCR errors and how can
they be avoided?
4. What particular feature of PCR enables you to produce large quantities of
the DNA section in which you're interested from as little as
one original cell?
Glossary:
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Amplification: producing
many DNA copies from one original DNA target.
Annealing: binding
of the primer to the template or test DNA.
Annealing temperature:
The temperature at which a length of single-strand (heat denatured) DNA will
anneal to a complementary strand.
Bases: Adenine (A),
cytosine (C), guanine (G) and thymine (T) are molecules that make up DNA. Bases
in DNA form two pairs of complementary molecules, hydrogen bonds can link adenine
to thymine and guanine to cytosine. If the sequence of bases matches a complementary
sequence in a second strand of DNA the bonds can hold the two strands together
(Watson-Crick pairing) and form double-stranded DNA.
Denaturing: The process
of separating double stranded DNA into single strands by breaking the hydrogen
bonds. This is most often accomplished by heating the DNA solution to temperatures
greater than 90ºC.
DNA Polymerase: An
enzyme that can synthesize new complementary DNA strands using a DNA template
and primer. Taq DNA polymerase is commonly used for PCR reactions.
DNA: Deoxyribonucleic
Acid (DNA) is the genetic material that is passed from parent to daughter cells.
DNA contains the following four nucleotides: dATP, dCTP, dTTP, and dGTP.
dNTPs: Deoxynucleotide
triphosphates (dNTPs) are the bases added to a primer during the PCR that comprise
the newly synthesized strand. Examples of dNTPs are dATP, dUTP, dCTP, dGTP and
dTTP.
Elongation or extension:
Phase of PCR cycle following annealing of primer during which the Taq polymerase
synthesizes a strand of DNA. The optimum temperature depends on the enzyme used
but is usually between 68-72? C.
Hybridization: The
formation of stable duplexes of two DNA complementary strands via Watson-Crick
base pairing (A pairs with T and G pairs with C).
Oligonucleotide (Oligo):
Short sequences of nucleotides that are used as primers for the PCR.
PCR: Polymerase chain
reaction. Amplification of specific lengths of DNA by repeated 'thermal cycling'
reactions using polymerase enzyme.
Primer: Short sequences
of DNA (oligonucleotides) that are complementary to sequences of the template
DNA. A molecule that initiates the synthesis of a larger molecule. They are
usually between 15 and 30 bases in length.
Taq polymerase: Thermus
aquaticus DNA polymerase used in PCR reaction to synthesize DNA. Is stable at
higher temperatures than other DNA polymerases. Taq polymerase was first isolate
from Thermus Aquaticus, a bacteria that lives in hot springs at temperatures
up to 75ºC. The DNA polymerase purified from this bacteria can survive
temperatures over 90ºC.
Template DNA: DNA from source that is the material to be amplified using
PCR.
Thermal Cycler: Programmable
temperature block that rapidly changes the temperatures to those needed for
PCR. A typical PCR may have to cycle at 94ºC then 55ºC and then 72ºC
and repeat this cycle for as many as 30 to 40 cycles.
5' and 3': The orientation
of nucleic acids is indicated by the terms 3' or 5'. These refer to structures
within the phosphate/sugar backbone of the molecules. The 5' end has a phosphate
group whereas the hydroxyl (OH) end of the molecule is designated 3'. Single
strand DNA sequences are normally written from the 5' end of the molecule to
3' end. In double-stranded DNA the lower, second strand will lie in the opposite
direction from 3' to 5'. Taq enzyme manufactures DNA from the 3' end of the
primer.
Interesting
web sites: top
The DNA Learning Center (produced by Cold Spring Harbor Laboratory)
The PCR Jump Station (scroll down to the "General Theory" section)
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