Naveen Sooknanan McGill Fall 2011
Molecular Genetic Techniques:
DNA Cloning, Polymerase Chain Reaction (PCR) and DNA Sequencing are the fundamental
techniques used in molecular biology. Other, more complex techniques borrow general aspects
from these 3 techniques.
For many reactions involving DNA in molecular biology, a large amount of DNA is
required, which can be difficult to obtain without the use of mainly DNA cloning and
PCR.
Plasmids are circular, double stranded DNA molecules (dsDNA) which are much smaller than
genes (usually 2000-10000 kbp)
Plasmids are the most common vectors used in DNA technology
o Vectors are media which carry specific genes of interest
They are extrachromosomal, meaning they are not part of the genome
They can be found in bacteria (mainly prokaryotes) and lower eukaryotes
The replication of plasmids occurs before cell division
The E. coli plasmid is a common vector used in labs.
The polylinker contains multiple endonuclease restriction sites
which scientists can cleave and genes of interest can be
inserted.
The amp gene codes for ampicillin resistance
o ampicillin is an antibiotic which normally kills E. coli
The ORI is the origin of replication for this plasmid (prokaryotes only have one origin)
DNA cloning involves the insertion of a desired DNA fragment into a plasmid and allowing the
replication procedure to take place, producing many more copies of the desired gene.
A restriction endonuclease cleaved the phosphodiester bonds
at the restriction site
o There is only one restriction site per endonuclease on
one plasmid
This breaks the circular plasmid into a linear DNA molecule
Although restriction endonucleases normally create cuts with
sticky ends, Sma1 is able to cleave straight through the DNA
molecule, creating blunt ends
The vector DNA molecule is then able to be attached to the plasma fragment with a
corresponding sticky ends
o This is carried out by an enzyme carried out by T4 DNA ligase using 2 ATP and
producing 2 AMP and 2 Pyrophosphate
This creates a recombinant plasmid (meaning it contains foreign DNA)
The recombinant plasmid is then mixed with E. coli and treated with CaCl a2d 42° C
heat pulses to make the cell membrane permeable, allowing the plasmid to enter the E.
coli cells (transformation)
The cultures are then placed on agar plates containing ampicillin which allows
differentiation of only the transformed cell
1Naveen Sooknanan McGill Fall 2011
o Wild type cells do not have ampicillin resistant plasmids die when they are placed
on the plate
o Transformed cells survive due to the presence of the Amp gene in the plasmids
Cell division and plasmid replication both increase the amount of plasmid vectors in the
colony
o These cells form a colony of recombinant cells with spatial limitations
o Plasmid replication is independent of chromosome replication
Plasmids nowadays are heavily engineered to replicate more quickly than naturally found
plasmids. Although they vaguely resemble naturally found plasmids, the plasmids commonly
used in labs are synthetic and cannot be found naturally.
Using this method, it is possible to create a library of DNA fragments which can all be
retrieved with one endonuclease
The polymerase chain reaction (PCR) is a very fast procedure of duplicating DNA and can be
done in 25 minutes to 2 hours, compares to the 3-4 days it takes for DNA cloning to take place.
PCR requires:
A DNA template to polymerize, part of genomic DNA
Oligonucleotide primers (DNA primers) which can be purchased given that the DNA
being polymerized is already known
Taq DNA polymerases, which are takes from a species which lives in extreme
temperature conditions
o Taq DNA polymerase can withstand the heat involved with PCR
dNTPs used for DNA synthesis
A thermal cycler which is capable of creating the heat patterns necessary in a PCR
reaction
An important aspect of DNA is its ability to denature when heated but more importantly, its
ability to renature when cooled back down. This is a fundamental principle exploited in PCR. A
cycle of PCR involves
First, heating the DNA to 95° to denature the DNA
o This heat will denature most DNA molecules
Second, cooling to 60° to anneal primers
o The unwound template due to the high
temperature allows for the free binding of
oligonucleotide primers
And third, heating to 72° to promote DNA extension
o dNTPs and DNA polymerases present in excess
to speed up the reaction
This cycle is repeated multiple times (usually 20-40
times) in order to increase the amount of DNA produced
exponentially
The synthesis of new duplexes proceeds very rapidly in PCR.
Starting with one duplex, approximately 2 duplexes are
produced after n cycles.
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