iGEM 101: Introduction to Synthetic Biology
Spring 2015
Session 6 – Polymerase Chain Reaction (PCR)
Overview: One of the most ubiquitous techniques in molecular biology is polymerase chain reaction (PCR).
While there are many variants of PCR and specialized applications, all have in common the exponential
amplification of a specific DNA sequence. A common use is to use PCR as a means of conformation, based on
the different sized products that result depending on what the composition is of the DNA sequence between the
two primers. This is the type of PCR that will be done to confirm that the plasmid that was finally assembled
last week includes its correct insert.
Recommended Reading:
Barker, At the Bench Chapter 6
PCR Components: PCR is effectively a form of in vitro DNA replication. As such, it requires a DNA
polymerase to add bases to the newly synthesized DNA strand, dNTPs to supply the bases being incorporated,
DNA template to replicate, a primer with a free 3' hydroxyl group to extend from, and the necessary cofactors
for the polymerase enzyme to function. The primers anneal to complimentary DNA strands, giving PCR its
sequence specificity.
Two primers are used in PCR, to amplify the sense DNA strand in one direction, and the antisense DNA strand
in the reverse direction. Proper primer design is crucial for reliable PCR. A length of around twenty bases
balances the needs for sequence specificity (improved by adding more complementary bases) and ease of
replication (improved by lowering the amount of bases to make it easier for the DNA strands to separate). Other
factors, such as secondary structures, repeat sequences, and homologies can also produce issues during PCR
amplification, as primers may mis-anneal or anneal incorrectly with themselves (forming primer dimers)
PCR Cycle: A thermocycler controls the precise series of temperature changes required for PCR. For primers to
bind, the sequence must first be made single stranded by denaturation at temperatures near the boiling point of
water. Cooling the reaction gives primers the opportunity to anneal, with primers having their maximum
specificity when the annealing temperature is customized based on the primer's base sequence. Multiple cycles
of extensions by the polymerase then synthesize DNA. The polymerases used in PCR are special in that they
work at very high temperatures. This preserves the specificity of the primers and keeps the DNA single stranded
during replication.
PCR Variants: PCR is a versatile technique that has been adapted for many specialized applications. For
example, qPCR quantifies the amount of DNA present during replication, which is then used as a measurement
of relative DNA levels at the reaction's start. Extension PCR allows one to add short sequences, such as
restriction sites, to the ends of an existing piece of DNA. A third example is reverse transcription PCR, which
starts with RNA and produces cDNA as a product.
PCR (Phusion)
Purpose: Amplify a specified DNA sequence
1. Make Master Solution
-Mix the following in a microcentrifuge tube
5x Phusion Reaction Buffer 5 µl
dNTP
0.5 µl
5’ Forward Primer
1 µl
3’ Reverse Primer
1 µl
Phusion Polymerase
0.3 µl (add last)
ddH2O
16 µl
*Optional addition of 0.25 µl DMSO
-Fill properly labeled PCR tube with approximately 23.8 µl of the Master Solution
2. Add Template DNA
-If necessary dilute template DNA into proper concentration for PCR
- Add 1 µl template DNA into each PCR tube, or 10-100 ng
3 Program thermocycler
-Initial denaturation at 98 °C for 30 seconds
-30 cycles of 98 °C for 10 seconds, followed by the primer-specific annealing temperature for 30
seconds, followed by 72 °C for 30 seconds per kilobase amplified
-Final extension at 72 °C for 5 minutes