PCR & DNA Profiling
6C.4 & 6C.5
Polymerase Chain Reaction (PCR)
• A laboratory technique used to amplify a specific target region of
DNA.
• E.g. a gene whose function a researcher wants to understand, or a
genetic marker used by forensic scientists to match crime scene DNA
with suspects.
• The purpose is to make enough of the target DNA region that it can
be analysed or used in some other way.
Polymerase
• Like DNA replication in an organism, PCR requires a DNA polymerase
enzyme that makes new strands of DNA, using existing strands as
templates.
Taq Polymerase
• High temperature is used repeatedly in PCR to separate the strands of
the template DNA, causing DNA polymerase to become denatured.
• The DNA polymerase typically used in PCR is called Taq polymerase,
after the heat-tolerant bacterium from which it was isolated (Thermus
aquaticus).
• T. aquaticus lives in hot springs and hydrothermal vents ∴ its DNA
polymerase is very heat-stable, with an optimum temperature of
around 70°C.
• This heat-stability makes Taq polymerase ideal for PCR.
PCR Primers
• Like other DNA polymerases, Taq polymerase can only make DNA if
it's given a primer, a short sequence of nucleotides that provides a
starting point for DNA synthesis.
• In a PCR reaction, the experimenter determines the region of DNA
that will be amplified by the primers.
• PCR primers are short pieces of single-stranded DNA, usually around
20 nucleotides in length.
• Two primers are used in each PCR reaction, and they are designed so
that they flank the target region.
• When the primers are bound to the template, they are extended by
the Taq polymerase, and the region between them will be copied.
The Steps of PCR
• Denaturation (96 °C): Heat the reaction strongly to separate, or
denature, the DNA strands. This provides single-stranded template for
the next step.
• Annealing (55 - 65°C): Cool the reaction so the primers can bind to
their complementary sequences on the single-stranded template
DNA.
• Extension (72 °C): Raise the reaction temperatures so Taq polymerase
extends the primers, synthesizing new strands of DNA.
• This cycle repeats 25 - 35 times in a
typical PCR reaction, which
generally takes 2 - 4 hours,
depending on the length of the
DNA region being copied.
• If the reaction is efficient, the
target region can go from just one
copy to billions.
• It’s not just the original DNA that’s used as a template each time, the
new DNA that’s made in one round can serve as a template in the
next round of DNA synthesis.
• The number of DNA molecules roughly doubles in each round of
cycling.
• Exponential growth
Gel Electrophoresis
• Used to visualise the results of PCR.
• Gel electrophoresis is a technique in which fragments of DNA are
pulled through a gel matrix by an electric current, and it separates
DNA fragments according to size.
• A standard, or DNA ladder, is typically included so that the size of the
fragments in the PCR sample can be determined.
• https://learn.genetics.utah.edu/content/labs/gel/
• DNA fragments of the same
length form a "band" on the
gel, which can be seen by eye
if the gel is stained with a
DNA-binding dye.
• For example, a PCR reaction
producing a 400 base pair (bp)
fragment would look like this
on a gel:
Applications of PCR
• Using PCR, a DNA sequence can be amplified millions or billions of
times, producing enough DNA copies to be analysed using other
techniques
• used for creating DNA profile for e.g. forensics or classification.
• used to amplify genes associated with genetic disorders from the DNA of
patients.
• used to test for a bacterium or DNA virus in a patient's body: if the pathogen
is present, it may be possible to amplify regions of its DNA from a blood or
tissue sample.
Sample problem: PCR in forensics
• You have just received a DNA sample from a hair left at a crime scene,
along with DNA samples from three possible suspects.
• Your job is to examine a particular genetic marker and see whether
any of the three suspects matches the hair DNA for this marker.
• The marker comes in two versions. One contains a single repeat, while
the other contains two copies of the repeat.
• In a PCR reaction with primers that flank the repeat region, the first
allele produces a 200 bp DNA fragment, while the second produces a
300 bp DNA fragment.
• You perform PCR on the four
DNA samples and visualize the
results by gel electrophoresis,
as shown.
• Which suspect's DNA matches
the DNA from the crime scene
at this marker?
Introns and Exons
• Functions?
• Which does DNA Profiling use?
• Micro-satellites: 2-6 bases repeated 5 to 100 times
• Mini-satellites: 10-100 bases repeated 50 to several hundred times
• Number of repeats linked to relatedness
• 11+ matching micro-satellites = same individual
