ADVANCED MOLECULAR
BIOLOGICAL TECHNIQUES
The Polymerase Chain Reaction (PCR)
Restriction Fragment Length Polymorphism
Sequencing of DNA bases
Transformation (By Emily Kennedy)
By Ruba Safieddine

Developed by Mullis, who was awarded the Noble
Prize in 1992

PCR is a process that allows the production of
many copies of DNA fragments

PCR uses heat (temperatures between 94 º C -96 º C) to
separate the 2 strands of DNA by breaking the Hbonds between complimentary base pairs

The two strands are used for replication

Temperature is lowered to 50 º C- 60 º C and DNA primers
attach to the DNA templates.

At 72 º C Taq polymerase identify the DNA primers and begin
to attach free nucleotides to complete the complimentary
strand of each template.

When the replication is complete, the process is repeated.
Since each new cycle has more templates that can be
used for replication, each cycle results in an “exponential
increase in the number of copies of the target DNA”
This process can result in 2 types of
strands:
Variable-length strands:
A mixture of strands of DNA that
have been replicated and are of
unequal length

Constant length strands:
A mixture of strands of DNA that
have been replicated and are of
equal length

Forensic criminal investigations:
a single cell can be replicated many times, therefore only a small
sample of DNA evidence is required from a crime scene

Medical Diagnosis:
to detect the presence of HIV

Genetic Research
uses fossil remains to determine if 2 species are closely related
Video of PCR: http://www.youtube.com/watch?v=_YgXcJ4nkQ&feature=related
 Polymorphism: any difference in DNA sequence that
can be detected between individuals (in the coding or
non coding region)
 can be used in coding regions to detect
specific mutations in an individual
e.g. Sickle cell anemia
 Polymorphic: a term used to describe the genomes of
individuals from the same species
RFLP analysis is the comparison of the differences in the length of DNA
fragments between different people using the following techniques:
 Step 1: DNA is extracted from sample and then digested by restriction
enzymes called endonucleases
 Step 2: The DNA is run on gel electrophoresis
where it appears as a blob
(since there are so many fragments and they are so close in size)
 Step 3: Double stranded DNA is placed in a denaturing solution causing it
to become single stranded. Using a process called southern blotting,
an electric current allows the DNA in the gel to transfer to a nylon
membrane.
 Step 4: The nylon membrane is put in a solution with radioactive nucleotide
probes that attach at specific locations.
 Step 5: Hybridization occurs
complimentary base pairing through the formation of hydrogen
bonds between the probes and the DNA
 Step 4: The nylon membrane is put in a solution with radioactive nucleotide
probes that attach at specific locations.
 Step 5: Hybridization occurs
complimentary base pairing through the formation of hydrogen
bonds between the probes and the DNA
 Step 6: The nylon membrane is placed against x-ray film where an
autoradiogram (gel pattern on the x-ray film) is formed
* This pattern can be used in forensic investigations to connect a suspect to a
crime scene based on a DNA match. It can also be used to find a mutation that
may cause a genetic disorder.
•
Diagram of RFLP : http://homepage.smc.edu/HGP/images/rflp.gif

determining the exact sequence of
nucleotide bases in a specific DNA
strand

Main method Dideoxy Sanger method
developed by Fred Sanger
in 1977
Dideoxy Sanger method:
-Four copies of a single stranded DNA with a radioactively labeled
primer on its end are placed in 4 reaction mixtures
-Each test tube contains DNA polymerase, all four deoxynucleoside
triphosphates (dNTP) e.g. Adenine (dATP) and one radioactively labeled
dideoxy analogue (either dideoxy-adenine (ddATP) , dideoxy-thymine (ddTTP), dideoxyguanine (ddGTP), or dideoxy-cytosine (ddCTP).
-A dideoxy analogue has a ribose sugar that lacks an –OH group on the
2’ and 3’ carbon
Therefore synthesis of a new DNA strand stops because the
next complementary base cannot be added by the DNA
polymerase
-Since only a small portion of the mixtures contain
dideoxy analogues, different length of
complimentary DNA will form depending on when
the ddNTP is integrated.
e.x.
3’ G G A C T A T C C A T T A G C 5’
Strands that may be produced:
5’ C C T G
C CTGATAG
C CTGATAG G
C C T G A T A G G T A A T C G 3’
Strand to be replicated is
placed in test tube with
dideoxy analogue Guanine
 The second the dideoxy
analogue is integrated the
chain is prevented from
growing any further
-Next, gel electrophoresis is used to separate the
strands of different length
-
Once this is done they are placed against an
X-ray film where the pattern can be determined
by method of autoradiogram
Video showing process:
http://www.youtube.com/watch?v=oYpllbI0qF8
 Transformation is the process of bringing in foreign DNA from
another source.
 This commonly occurs in bacterial cells when a plasmid or a
virus is inserted which changes or introduces a new
characteristic to the bacteria cell.
 The plasmid or the virus is known as a vector, which carries the
DNA into the host cell.
 Once the bacterium takes this foreign DNA it is called a
competent cell. But not all bacteria will take this new DNA, so
transformation does depends on the cell being competent.
 Although, cells can be encouraged to take this DNA and it
is through working with the charges of the ions and
working with the ranges of temperatures. This is done by:
-Having DNA and cell membranes with both slightly
negative charges
-When the plasmid with the DNA and the bacteria is put
in CaCl2 solution, the positive calcium ions will attract to
the negative charged plasmid which is give a neutral
charge.
-By having the solution at a low temperature and then
dramatically and rapidly increasing it, a draft is created
that allows the plasmid to move through the pores of the
cell membrane and into the cytoplasm.
To see if transformation did successfully occur you can
perform a test that is illustrated at this website:
http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/ima
ges/bacterial_transformation/4063-1-eng AU/bacterial_transformation_large.jpg
Basically, this test uses the principle of comparing your samples.
 First have a Petri dish of the bacteria that does not have the plasmid
containing the genetic information.
 Then compare it to a Petri dish of your sample of bacteria that you
encouraged transformation.
 Depending on what the plasmid or virus’s DNA information was, it will
be different from your original bacteria.
 Let’s say the plasmid contains an antibiotic in the genetic
code of the DNA, your second Petri dish should have a
noticeable decrease in surface area of the bacteria.
 If there is a decrease, transformation was successful. *Of
course, not all of the bacteria should have disappeared
because not every bacteria cell was transformed.
 More recently, electric shock has encouraged
transformation in plant cells through electroporators. By
sending electric shocks, the strong cell walls loosen and it
becomes easier for the foreign DNA to get through.
 They can also do this by using a gene gun to break the ridge
walls. They do this by:
-Taking a water droplet and charging it.
-Send it through a tube with an electrical discharge.
-At the end of the tube is a sheet that has DNA wrapped in
gold particles. They use gold because it is very nonreactive.
-Then the water droplet that contains these DNA wrapped
gold particles is propelled at the cells and the force of the
impacts breaks the cell walls and cell membranes.
Di Giuseppe, Maurice, Vavitsas, A., Ritter, Dr. Bob, & F.,
Douglas (2003). Biology 12. Canada: Thomson Nelson.
* All definitions are directly from the text*