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Polymerase Chain Reaction (PCR) Field Trip
Background Information
The Polymerase Chain Reaction (PCR) is a widely used technique
for copying DNA. Starting with only a small sample of DNA, PCR can
generate many copies of a specific DNA segment to be used for
further analysis. This process is also called DNA amplification. PCR
has revolutionized molecular biology, and is now routinely used in
biological research, forensics (criminal investigations), medical
testing, and anthropology. During the PCR field trip, students
set-up and perform a PCR reaction, learn the underlying principles
of PCR, and analyze their results on an agarose gel.
In 1983 Kary Mullis, a scientist at the Cetus Corporation in
California, imagined a way to replicate (copy) DNA in the lab. He
worked on the idea for two years, and in 1985 published and filed a
patent for his idea. In 1993 he won the Nobel Prize in Chemistry for
his work.
PCR utilizes:
· template DNA - the starting DNA of interest.
· two primers - short, single-stranded, synthesized
pieces of DNA that complement sequences on each side of the
region of the template DNA that is being amplified.
· Thermostable DNA polymerase - typically Taq
(Thermus aquaticus), a heat stable enzyme capable of
adding nucleotides to a growing DNA strand.
· dNTPs - a supply of the 4 nucleotides needed to
make the new DNA strands.
· magnesium - a cofactor for the polymerase.
· a buffer solution - to maintain the pH and salt
concentrations appropriate for the polymerase
Once these components are combined they go through a series of
temperature changes (cycles), repeatedly, in a machine called a
thermocycler. This process will generate exponential copies of the
DNA segment of interest. In other words, if you start with 2 copies
of the DNA segment of interest, after 20 cycles you will
theoretically have 2(nth) = 2(20th) = 1,000,000 copies of that
segment.
Each cycle consists of three parts: denaturation (D), annealing
(A), and elongation or extension (E). Denaturation separates the
double-strands of the DNA molecule at a relatively high temperature
of 90-96°C, annealing allows the primer
sequences to match and bind to the flanking regions of the target
area at a moderate temperature between 40-70°C,
and elongation or extension occurs as the polymerase adds
nucleotides to the growing strand at 68-72°C.
The initial denaturation may last from 2-5 minutes, then is
typically 30 seconds during subsequent cycles. Annealing and
elongation steps are typically 1 minute each, with a final
elongation that may last up to 10 minutes. After the cycling is
complete the PCR product is held at 4°C.
The number of cycles, temperatures and time lengths are programmed
into the thermocycler, which is somewhat like a computerized heat
block.
Since the anticipated product length is known, PCR products can
be evaluated using an agarose gel when run alongside a DNA size
standard, or marker, with DNA bands of known sizes. Template DNA
will be longer in length then the desired PCR product, run more
slowly through the gel, and appear as a band closer to the top of
the gel. Unbound primers or “primer dimers” will be shorter, run
faster, and create smears of DNA closer to the bottom of the gel.
The amplified DNA in the PCR field trip ranges in size from 180 to
1500 base pairs in length, the primers are 22 and 24 base pairs in
length.
PCR has become part of the popular culture. “Jurassic Park”
and “CSI” are just two examples where PCR is crucial to the
plot. Some fun PCR facts to share with your students*: …PCR has
been used to amplify DNA from…
· poached moose meat in hamburger
· a preserved quagga (an zebra relative that became
extinct 100 years ago)
· crime scenes
· eight-celled human preembryos, to detect cystic
fibrosis
· the brain of a 7000 year old American mummy
· patients for disease diagnosis
*Life third edition, Ricki Lewis, WCB
McGraw-Hill, 1998.
You can also find some useful information on the web about PCR,
including animated computer tutorials. One tutorial sponsored by
Cold Spring Laboratory can be found at:
www.dnalc.org/shockwave/pcranwhole.html.
Please note that this requires a Shockwave Plug-in to view, you will
be prompted on how to download this plug-in when you visit the site.
The PCR Field Trip includes three major components; students set
up a PCR reaction, while the reaction is in the thermocycler they
learn the theory behind PCR, after the reaction is finished, they
analyze their PCR products on an agarose gel. They will also receive
a photocopy of their gel to take with them. During this field trip,
students learn about a variety of techniques including; pipetting,
using a centrifuge, making an agarose gel, running and analyzing the
gel.
If you have any questions about this field trip, please give us a
call, or bring your questions along and we can discuss them during
the lab. Thank you for your interest in BTCI's Biotechnology Field
Trips program.
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