Name:

Abby Yong Sze Yee (14WAR03484)

Wee Sze Ping (14WAR05085)
Wong Yu Ning (14WAU06263)

Programme: Bachelor of Science (Honours) in Bioscience with Chemistry (RBS 3 Group 2)

Course:

BABS3213 Techniques in Biotechnology

Date:

8 August 2016

EXPERIMENT 5

Title: Polymerase Chain Reaction (PCR) of lambda DNA with the use of FIREPol DNA
Polymerase

Aim:
The amplification of lambda DNA by Polymerase Chain Reaction (PCR) method with the use
of FIREPol DNA Polymerase to further understand the principles and applications of the
PCR method.

Introduction
Polymerase Chain Reaction (PCR) is a system for DNA replication that allows a desired
DNA sequence to be selectively amplified, or enriched, several million-fold. Within a
dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end
result is a copy of the entire genome. Within the PCR, one indispensable enzyme; namely the
DNA polymerase, is used to amplify a specific fraction of the genome. During cellular DNA
replication, enzymes first unwind and denature the DNA double helix into single strands.
Then, RNA polymerase synthesizes a short stretch of RNA complementary to one of the

DNA strands at the start site of replication. This DNA/RNA heteroduplex acts as a "priming
site" for the attachment of the DNA polymerase, which then produces the complementary
DNA (cDNA) strand (Custom Essay 2015). [1]
In this experiment, PCR is carried out on a lambda DNA. Lambda is a medium size E.coli
bacteriophage. The DNA molecule of 48502 basepairs is linear and except for the extreme
ends double-stranded. At each end the 5' strand overhangs the 3' strand by 12 bases. The
sequences of the ends are complementary. At ambient temperatures, in a solution containing
purified Lambda-DNA these 'cos ends' may pair and form the 'cos-site'. As a consequence, the
DNA is partly circularised or have formed concatemers (Bioinformatics 2016). [2]
There are three main steps in the PCR cycle; namely, Denaturation, Annealing and Extension.
During denaturation, the DNA is denatured into single strands at 94-96 oC for one minute. In
annealing, the primers hybridize to their complimentary sequences on either side of the target
sequence at 50-65oC for 40 seconds. The extension step occurs at 72 oC and is where the
polymerase binds and extends a cDNA strand from the primer. As amplification proceeds, the
DNA sequence between the primers doubles after each cycle. Following thirty such cycles, a
theoretical amplification factor of one billion is attained (Cold Spring Harbor Laboratory
2015). [3]

Source: Microbiology Info [4]

References
[1] Custom Essay 2015, Lambda Dna Amplification By Polymerase Chain Reaction (Pcr),
viewed 17 August 2016, < http://custom-essay-cheap.com/lambda-dna-amplification-bypolymerase-chain-reaction-pcr/>.
[2]

Bioinformatics

2016,

About

Lambda

DNA,

viewed

17 August

2016,

<

http://www.bioinformatics.nl/molbi/SCLResources/lambda.htm>.
[3] Cold Spring Harbor Laboratory 2015, Amplifying Lambda DNA by Polymerase Chain
Reaction

(PCR),

viewed

17

August

2016,

<

http://labprotocols.dnalc.org/files/046_amplifying_lambda_DNA.pdf>.
[4] Microbiology Info, Polymerase Chain Reaction (PCR)- Principle, Procedure, Types,
Applications

and

Animation,

viewed

17

August

<http://www.microbiologyinfo.com/polymerase-chain-reaction-pcr-principle-proceduretypes-applications-and-animation/>.

2016,