PCR Script & Vocabulary Sheet
PCR Script
When students use the script below while they manipulate the PCR components it will reinforce
their understanding of the PCR Cycle.
Script for each step of the To do and notice section:
[Step 1] Say: “The PCR mixture is started in the thermocycler at 25C. The temperature begins
to rise to 95C and the first PCR cycle begins. The template DNA separates (denatures)
into two template strands.”
[Step 2] Say: “The mixture is cooled to about 50C. The 3-letter primer TGT attaches (anneals) onto
each template strand following Chargaff’s Rule (Adenine bonding with Thymine, Guanine
bonding with Cytosine)”
[Step 3] Say: “The mixture temperature is raised to 72C. The enzyme DNA polymerase begins
attaching nitrogenous bases (A’s, T’s, C’s, G’s) onto the template strands starting from
the primers and extending towards the longer end of each template. This process is
called polymerization.”
[Step 4] Say: “PCR cycle 1 is complete. Cycle 2 begins when the thermocycler again raises the
mixture temperature to 95C. The new DNA pairs denature, forming 4 strands. Two of
the strands contain both a primer and a polymerized extension.”
[Step 5] Say: “The temperature cools again to 50C and primers anneal to the strands. Then the
mixture is heated to 72C and DNA polymerase again polymerizes (extends) the
strands.
[Step 7, no script for step 6] Say: “How many amplified (copied) products (DNA molecules) are
there? How many of the products have hanging ends? How many do not have hanging
ends? The pairs with hanging ends are called long products. Those without hanging
ends are called short products. The short products are also called target sequences.
They are exact copies of the portion of template DNA needed by the scientist and are
the goal of PCR.”
PCR Vocabulary
Anneal: The binding of DNA primers and nitrogenous bases onto a template DNA strand at
temperatures of 37 - 70C.
Chargaff’s Rule: The rule of base pair relationships in DNA, based on typical molar
concentrations of molecules called purines and pyrimidines occurring in the DNA molecule. The
nitrogenous bases adenine and guanine are purines while thymine and cytosine are pyrimidines.
The rule explains that purines always bond with pyrimidines. Specifically, adenine always bonds
with thymine and cytosine with guanine. These base-pair bonds hold the two strands of DNA
together and give DNA its molecular stability.
PCR Cycle Models - PCR Script & Vocabulary Sheet
© 2013, RAFT
Denaturation: Loss of activity of an enzyme or nucleic acid molecule as a result of structural
changes induced by heat or other means. The heating of the PCR mixture denatures the DNA
molecule where the two strands bond and hence the strands separate.
DNA polymerase: The enzyme responsible for attaching nitrogenous bases onto template DNA
during replication both in cells and in a PCR mixture.
DNA replication: The common way in which DNA is synthesized. Each of the strands in a
double-helix acts as a template for a new strand. Hence, after replication, each double helix
consists of one old and one new strand. This is why this process is called semi-conservative
replication. This process is controlled through cellular chemistry in organisms while in PCR it is
controlled through variation in temperature.
Enzymes: A protein produced by a living organism that acts as a catalyst to bring about specific
biochemical reactions. As catalysts, enzymes lower the amount of energy required to start the
reactions, allowing the reactions to occur much faster. Each enzyme is specific to a particular
biochemical reaction, e.g. DNA polymerase builds DNA polymers by speeding up base-pairing
reactions, attaching only bases to a DNA strand.
Long products: Replicated, double-stranded DNA molecules having a section on one strand
containing nitrogenous bases that are not bonded to their complimentary bases. This section is
often termed a “hanging” or “sticky” end. As a function of the number of PCR cycles, long
products are represented as f(n) = 2n, where n equals the number of cycles.
Nitrogenous bases: The purines or pyrimidines in nucleic acids attached each sugar in a DNA
strand (see Chargaff’s rule). Adenine, thymine, guanine, and cytosine are symbolized as A, T,
G, and C, respectively.
Primer: A short, single-stranded DNA segment that is the necessary starting material for the
synthesis of a new DNA strand. For purposes of PCR, the segments are engineered in a
laboratory.
Polymerization: The linking of monomers (single molecules) to generate polymers, which are
long molecule chains. Examples include proteins, sugars, and DNA. In DNA, the monomers
linked together by DNA polymerase are the nitrogenous bases adenine, thymine, cytosine, and
guanine forming the DNA polymer (sequence).
Short products: The DNA sequences of interest to scientists using the PCR technique. They
are replicated segments of double-stranded DNA based on the original DNA sample (template).
Short products are mathematically represented as a function of the number of PCR cycles as f(n)
= 2n – 2n, where n equals the number of cycles.
Target sequence: A portion of a DNA sample of interest to biologists or forensic scientists. This
is also called the short product derived through PCR.
Template: A molecule or surface upon which another molecule is synthesized in complimentary
fashion, as in the replication of DNA.
Thermocycler: A machine into which mixtures of DNA, nitrogenous bases, DNA polymerase,
and primers are added for the purpose of PCR. The machine cycles the mixture through specific
high and low temperatures, triggering each step of the PCR process until the desired number of
target sequence copies is achieved.
PCR Cycle Models - PCR Script & Vocabulary Sheet
© 2013, RAFT