BIOS390 Eric Kyle
BIOS 390 Week 1 LAB 1
LAB 1: Polymerase chain reaction
Polymerase chain reaction (PCR) is a laboratory technique used to increase, or make many
copies of, a specific target region of DNA in vitro. The goal of PCR is to make enough of the
target DNA region that it can be analyzed or used in some other way. For example, DNA
amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a
plasmid for further experiments.
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands
as templates. 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 and is most active
around 70°C (a temperature at which a human or E. coli DNA polymerase would be
nonfunctional). This heat-stability makes Taq polymerase ideal for PCR because high
temperatures are used repeatedly in PCR to denature the template DNA, or separate its strands.
For tag polymerase to make DNA it must first be given a primer, a short sequence of nucleotides
that provide a starting point for DNA synthesis. The primer is a short piece of single-stranded
DNA determined by the scientist from a certain region of DNA. Two primers are needed in each
PCR reaction and they are given sequences that will make them bind to opposite strands of the
DNA template by complementary base pairing. Once the primers are fixed to the template, they
can be extended by the polymerase, and the region that lies between them will get copied.
The main ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and
nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that
allow DNA to be synthesized.
The basic steps are:
1. Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next step.
2. Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.
3. 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. When successful, the target region can
go from just one or two copies to billions of copies of DNA. There are many copies of the
BIOS 390 Week 1 LAB 1
LAB 1: Polymerase chain reaction
Polymerase chain reaction (PCR) is a laboratory technique used to increase, or make many
copies of, a specific target region of DNA in vitro. The goal of PCR is to make enough of the
target DNA region that it can be analyzed or used in some other way. For example, DNA
amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a
plasmid for further experiments.
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands
as templates. 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 and is most active
around 70°C (a temperature at which a human or E. coli DNA polymerase would be
nonfunctional). This heat-stability makes Taq polymerase ideal for PCR because high
temperatures are used repeatedly in PCR to denature the template DNA, or separate its strands.
For tag polymerase to make DNA it must first be given a primer, a short sequence of nucleotides
that provide a starting point for DNA synthesis. The primer is a short piece of single-stranded
DNA determined by the scientist from a certain region of DNA. Two primers are needed in each
PCR reaction and they are given sequences that will make them bind to opposite strands of the
DNA template by complementary base pairing. Once the primers are fixed to the template, they
can be extended by the polymerase, and the region that lies between them will get copied.
The main ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and
nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that
allow DNA to be synthesized.
The basic steps are:
1. Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next step.
2. Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.
3. 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. When successful, the target region can
go from just one or two copies to billions of copies of DNA. There are many copies of the
