PCR-RFLP Report

Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction is a method that uses the capability of DNA polymerase to
synthesize to new DNA strands which are matching to the template strand. A primer needs to be added
to the first nucleotide due to the fact that DNA polymerase only can add a nucleotide only onto a 3'-OH
group that already exists. Because of this condition, we are able to define a chosen region of template
sequence which we can then generate millions to billions of copies. This technique was developed by
Kary Mullis in 1983 and is a very common indispensable technique which has a variety of uses such as
DNA cloning for sequencing, genetic fingerprints, detection and diagnosis of infectious disease (often
cancer), etc.

Figure 1

Procedure:
PCR consist of 20-40 cycles of repeated temperature change and each cycle has mostly 3 distinct
temperature steps. The temperature used and the duration of treatment in each cycle depends upon
many factors such as melting temperature of the primers, etc. The cycle usually starts by a high
temperature of greater than 90 degree Celsius and ends with the same treatment as the initial step.
Starting Step: The reaction is heated to a temperature between 94-96 Degrees Celsius or 98 Degrees
Celsius if polymerase which can withstand higher temperatures. This continues for 1-9 minutes. (This
step is only required of the DNA polymerase requires heat activation)
Denaturing Step: This is the first step of the cycle. This step requires melting of the DNA template by
disturbance of hydrogen bonds between complementary bases which the resultant being a single-strand
of DNA molecules. To achieve this, the reaction must be heated to 94-98 Degrees Celsius for about 20 to
30 seconds.

Annealing Steps: Annealing must take place in order for the hybridization of primers to the single
stranded DNA template. This temperature must be high enough in order for the hybridizations to bind
specifically and low enough that it is possible for hybridization of primers to occur. Annealing
temperature is usually 3-5 Degrees Celsius lower than the melting temperature of the primers.
Extension/elongation step: This step consists of DNA polymerase synthesizing with a new DNA strand
which is complementary to the DNA template strand. This is done by adding dNTP's which complement
the template in the 5'-3' strand, condensing the 5'-Phosphate group of the dNTP along with the 3'hydroxyl group at the end of the nascent extending DNA strand. The temperature at which the reaction
is treated depends upon the primer used. The duration of this step also depends on the DNA
polymerase used also, and the length of DNA fragment used. Under optimal required temperature of
the primer, the DNA polymerase will polymerize a thousand base pairs per minute.
Final elongation: After the last PCR cycle is completed, the reaction is cooled to a temperature of 70-74
Degrees Celsius (optimal temperature needed for activity for most polymerases used in PCR) for 5-15
minutes to ensure that remaining single-stranded DNA is fully extended.
Final Hold (optional): Cooled to a temperature of 4-15 Degrees Celsius for short term storage of the
reaction.
To see if PCR has successfully generated expected DNA fragments, Agarose Gel Electrophoresis is used
for size separation of the PCR products.

Restriction
Fragment
Polymorphism (RFLP)

Length

Restriction
Fragment
Length
Polymorphism (RFLP) is a technique
that uses the difference in homologous
DNA sequences that can be detected
the presence of fragments which are of
different lengths after digestion of the
DNA samples. Because this method
acts as a molecular marker, RFLP is
specific to a combination of
clone/restriction enzyme. Double
stranded DNA is cleaved by hydrolyzing
the support which is between the
Deoxyribose sugar and the phosphate
the 5' phosphate and the 3'OH, this is
done by the restriction enzymes.
Loosely bound initially, these enzymes
bind to the DNA and move along the
path until a recognition sequence has
come in contact. symmetrical The

by

at

Figure 2

enzyme is able to cleave the DNA when

the tighter binding with the recognition
sequence leads to a symmetrical change.
Because there are many polymorphisms
in the genome, it is possible that some of
the variations end up occurring at the
recognition sequences for restriction
enzymes. If a sequence such as GAATTC
would normally be recognized by an
enzyme, or conversely, a non-cleavage
sequence could be changed to a
recognition sequence, making the
enzyme cut the DNA. The different
cleavage sites lead to differences in
length of the DNA segments after
digestion. Digested DNA can display a
detectable difference in bands from two
individuals if it is run through gel Figure 3
electrophoresis. This information can
then be used in a wide variety of situations.
Question: What causes Sickle Cell Anemia?
Sickle Cell anemia is a common type of sickle
cell disease which makes the body produce red
blood cells in the shape of a sickle, "Sickle
shape " intimates that the red blood cells are
molded like a bow. Ordinary red blood cells
molded and look like disks and are more
effective in moving through the veins. Red
blood cells contain an iron-rich protein called
hemoglobin. This protein carries the oxygen
and delivers it to any part of the body.
However, Sickle cells contain bizarre
hemoglobin called sickle hemoglobin or
hemoglobin S which is the cause to its peculiar
shape. Sickle cells are firm and sticky and have
a tendency to stop circulation system in the
veins of the organs and limbs. Blocked blood
stream can result in excruciating pain and
correspondingly can cause organ harm. It
likewise raises the danger of getting an
infection.

Sickle cell is the effect of a change in the #6 amino acid of the -globin chain of hemoglobin. Particularly
glutamic acid is changed over to valine, As seen in Figure 5, the gene of an individual with a normal
genome (because large percent of the human genetic code is similar to each other, so we can identify
which letter in the code is abnormal) compared to another individual with Sickle Cell Anemia. This
results from a change in the nucleotide A to T. This change kills a site perceived by the limitation
compound DdeI and is called a missense-severe mutation .The restriction enzyme in this would be Ddel
(sequence: 5'-GTNAG-3') and the probe will be a fragment - globin coding sequence. Using RFLP, we
can identify the mutation in genetic code. Figure 4
Because Sickle Cell Anemia is a genetic disorder
which relies only on one letter of the
sequence, the procedure is simplified as we
only need to test the beta-globin gene.
When both the DNA strands are compared,
the bar for 6th amino acid will have a
different blot showing. Hence, we can
Figure 5
conclude that Sickle Cell Anemia is caused
by a missense-severe mutation in the 6th
amino acid of the -globin chain of hemoglobin.

Biography
http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
http://en.wikipedia.org/wiki/Polymerase_chain_reaction
*(http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism#Analysis_technique)
http://www.nhlbi.nih.gov/health/health-topics/topics/sca/
http://upload.wikimedia.org/wikipedia/commons/9/96/Polymerase_chain_reaction.svg
http://www.ndsu.edu/pubweb/~mcclean/plsc431/markers/marker1.htm
Images
http://openwetware.org/images/3/35/Pierce_18_19_large_2.jpg (Figure 1)
http://homepage.smc.edu/hgp/images/rflp.gif (Figure 2)
http://upload.wikimedia.org/wikipedia/commons/d/d0/Roland_Gel.JPG (Figure 3)
http://www.nhlbi.nih.gov/health/health-topics/images/sickle_cell_01.jpg (Figure 4)
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SickleMutation.gif (Figure 5)