How does
PCR work?
The polymerase chain reaction
(PCR) is a method to find the genes/DNA
that make a particular plant genetically
modified. The goal is to amplify specific
sequences of DNA, for example the inserted
herbicide gene. DNA is amplified by
using specific short DNA sequences (called
primers) that flank the region
of the gene that is to be amplified.
In other words, the primers allow us
to find the "gene of interest"
by latching onto the gene. At the onset
of every PCR reaction we add DNA extracted
(DNA extraction)
from the GMO crop, primers, DNA polymerase
and bases to a vial.
Remember from our biology
classes that DNA is made up of two tightly
bonded strands, turning in a helix.
For our gene specific primers
to work, we need to open or separate
this DNA. A PCR thermocycler is a machine
that heats the DNA helix so it opens
(denatures). Once the DNA is
separated into two strands, the temperature
is dropped rapidly so that the primers
can latch (hybridize) onto
a single, complementary strand of DNA.
This is the annealing step.
The temperature of the thermocycler
is raised again so that the DNA polymerase
(an enzyme) produces more DNA.
This is the extension step.
A typical PCR program consists of 30-45
cycles of denaturing, annealing,
and hybridizing steps. We then
have enough of the gene/DNA synthesized
for analysis.
Analysis: After
the PCR reaction, the DNA from each
vial is carefully pipetted onto a agarose
gel. The gel is already stained with
ethidium bromide so that the added DNA
lights up under UV light. The charged
DNA molecules "run" down the
agarose gel and separate according to
their size. For example, large DNA fragments
stay on top of the gel, while smaller
DNA fragments travel the bottom of the
gel. A GMO plant would show a particular
DNA fragment indicative of the gene,
while a non-GMO plant would not show
that DNA fragment on the gel. Please
see a picture of a gel below: