Biochem 121.

1 Biochemistry of the Gene Laboratory

Bacteria and Blood DNA Extraction

Lopez, Richard; Palmario, Marijyke Katrina Patricia; Tan, Elaine

Group 2, Section MCD
March 14, 2011
Professor Joanne del Rosario

I. Abstract
Genomic DNA extraction from bacteria and whole blood was performed in
this experiment. For bacterial genomic DNA extraction, the method adapted from
Tillett and Neilan was used. Mainly, the method was composed of obtaining the
lysate, salting out and selective precipitation. For genomic DNA extraction from
whole blood, the Wang’s Lysis Method was used. The method consisted of lysis
to remove the erythrocytes which did not contain nuclei, salting out and selective
precipitation, as well. Both methods are rapid, efficient, safe and can serve as
standard laboratory protocols. Analysis using gel electrophoresis in 1x TAE
viewed under UV light is also presented in the paper.

II. Keywords

DNA Extraction, Bacterial Genomic DNA, Blood Genomic DNA, Tillett and
Neilan Method, Wang’s Lysis Method
_____________________________________________________________________

III. Introduction As for prokaryotes, specifically

The total genetic information of
an organism is specified by its
genomic DNA. In almost all
organisms, including prokaryotes
and eukaryotes, it is the DNA
which comprises the genome.
Viruses are the only exception to
this fact because they have RNA
genomes.
Since it constitutes the total
genetic information, genomic DNA
is generally large. In addition to
this, complexes of DNA-protein
are organized in chromosomes.
These characteristics, such as the
size of the genomic DNA, its
nature, and the number of
chromosomes vary from organism
to organism.
bacteria, chromosome is single in
number and circular in shape. On
the other hand, as for eukaryotic
organisms, chromosome is linear in
shape and is multiple in number.
Aside from chromosomes, one
can also consider the number of
base pairs of an organism as
distinct characteristics. For
example, the Lambda phage has
48, 502 base pairs. Man, Homo
sapiens, has approximately 3.3 x
109 and corn, Zea mays, has 3.9 x
109 base pairs. The molecular
weight of the genome, measured in
Daltons, is also important in DNA
analyses.
Today, DNA of organisms is
usually extracted because it is an
invaluable tool in the fields of

1
Molecular Biology, Biochemistry
and Forensic Analysis. However,
the process does not start at
extraction or isolation alone. It
actually starts on sample storage
before the isolation of genomic
DNA. Indeed, this is the most
crucial step since it is the quality of
the starting material which will
affect the quality and yield of the
isolated DNA.
For bacteria, sample storage is
relatively simple. Cold storage, at a
temperature range of -20 0C to - 80
0
C, is sufficient. However, DNA
extraction from animal tissues,
especially blood, poses several
challenges.
In blood DNA extraction, a
problem encountered is that
mammalian erythrocytes or red
blood cells do not contain nuclei.
Consequently, genomic DNA
cannot be extracted from RBC and
these must therefore be eliminated.
Upon elimination of erythrocytes
which comprise majority of the
blood sample, the leukocytes which
contain nuclei remain. Hence,
genomic DNA can be extracted
from blood.
For the isolation proper, DNA
can be separated from cellular
components using four main steps,
namely disruption, lysis, removal
of proteins and contaminants, and
then recovery of DNA.
In general, there are several
methods available for the
extraction of DNA. However,
regardless of the methods used, the
five main steps which include
storage, disruption, lysis, removal
of contaminants and recovery of
DNA still hold true in separating
DNA from cellular components.
In laboratories, there are seven
common protocols used for DNA
extraction. Each method has its
advantages and disadvantages, as
will be discussed here.
The first method is termed as
the preparation of crude lysates.
Here, the cell lysate is simply
incubated at high temperatures or
digested with proteinase K. The
recovered product will then be an
impure and contaminated genomic
DNA. This technique is not
advisable since it only gives rise to
high failure rates.
Another technique is the
salting-out method. This method is
actually a refinement of the crude
lysate. Here, the proteins and
contaminants are precipitated from
the cell lysate using high salt
concentrations. Ammonium acetate
and potassium acetate are usually
used as salts. However, DNA yield
and purity are not definitive in this
technique.
Organic extraction methods are
also available for DNA extraction.
Here, lysis is performed by the use
of a detergent, Afterwards, the
lysate is mixed with isoamyl
alcohol, polar and non-polar
reagents. The polar reagent is
usually phenol and the non-polar
reagent is usually chloroform.
Upon mixing, the components are
then separated into two phases –
organic and aqueous. Contaminants
are in the organic phase and DNA
molecules are in the aqueous phase.
However, both phenol and
chloroform may leave residues in
the DNA sample. More
importantly, these reagents pose
health hazards.
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 The principle behind that of the
organic extraction method is the
same as that of the CTAB method
which was not performed in class
due to the aforementioned
disadvantages.
In the Cesium Chloride Density
Gradient Method, the cell lysate is
first precipitated using alcohol.
Upon doing so, resuspended DNA
is mixed with both CsCl and EtBr,
then centrifuged. The DNA band is
then collected and then washed
with alcohol to remove EtBr. Not
only is this method tedious and
expensive, it also requires EtBr
which is a known mutagen.
The sixth method is the anion-
exchange method. In this method,
the phosphates of the nucleic acids
which have a negative charge
adhere to the positive charged
surface molecules on the substrate.
Using medium-salt washing, the
impurities are washed out and
DNA can be eluted using a high-
salt buffer. The problem with this
method is its high cost.
Lastly, silica-based methods
such as kits can be utilized. Here,
nucleic acids selectively adsorb on
the silica-gel membrane, in the
presence of high concentrations of
salts. Several buffers also ensure
that contaminants do not adsorb on
the membrane. DNA can then be
finally eluted out of the membrane.
As easy as it may seem, this
method is expensive to use in
undergraduate laboratory courses.
The methods discussed above
are some of the ones used in
genomic DNA extraction from
bacteria.
In animal tissues, such as
blood, the Wang’s Method can be
used due to its several advantages.
The Wang’s Method was first
published in 1994. It was
established in order to have a
standard protocol for rapid and
efficient DNA analysis. At that
time, extraction of DNA either
heavily relied on toxic reagents
such as phenol and chloroform, or
on tedious manipulations such as
literally spooling DNA.
The Wang’s Method is
characterized not only by its
rapidity and efficiency but also by
its reproducibility, high yield and
high purity.
Figure 1. The figure shows the
reproducibility of the method with
different sample volumes (Wang,
et. al., 1994).
In the fields of Biochemistry
and Molecular Biology, DNA
recovered from the said method
can be used for electrophoresis
since a high molecular weight is
preserved, for restriction
endonuclease digestion and for
PCR template.
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extraction from bacteria is different
from that of the DNA extraction from
animal tissues, specifically blood. The
two methods are described below.
Figure 2. Electrophoresis of
Purified Genomic DNA from
Human Whole Blood by Wang’s
Method and of its Digests from
Restriction Endonucleases (Wang,
et. al., 1994).
As can be seen in Figure 2,
Lane 1 serves as the marker for
HindIII digest. Lane 2 contains the
recovered genomic DNA which
shows desirable molecular weight.
Lanes 3 to 5 contain its digests
from BamHI, EcoRI and HindIII.
The figure supports the claim that
Wang’s method can indeed be used
for several applications involving
purified DNA.
In this paper, the extraction of
genomic DNA from bacteria and
animal tissue, specifically blood,
will be discussed. Also, in this
paper, the methodology used in
bacterial DNA extraction is the
Xanthogenate-SDS Method (Tillet
and Neilan, 2000). In blood DNA
extraction, the Wang’s Lysis
Method (Lu Wang, et. al., 1994) is
used. Upon discussion of the
extraction procedure, results and
qualitative analysis of the
recovered DNA will be presented.
IV. Methodology
The method used for DNA
A. DNA Extraction from Bacteria
The procedure for DNA
extraction from bacteria, called
Xanthogenate-SDS Method, is
adapted from Tillet and Neilan,
2000. In this method, the TER
buffer, XS buffer, isopropyl
alcohol, 70% ethanol and TE buffer
were the primary reagents used.
The TER buffer is composed of
10 mM Tris-HCl at pH 7.4, 1 mM
EDTA at pH 8.0 and 100 ug/mL
RNAse A.
The XS buffer is composed of
1% potassium ethyl xanthogenate,
100 mM Tris-HCl at pH 7.4, 1%
Sodium Dodecyl Sulfate and 800
mM ammonium acetate.
Lastly, the TE buffer was
maintained at a pH of 8.0.
As for the procedure, 10-20 mg
sample was first placed in a 1.5 mL
microcentrifuge tube. Afterwards,
it was re-suspended in 50 uL TER
buffer. 750 uL of freshly prepared
XS buffer was then added to the
tube. Inversion was done several
times to mix the components.
For 60 minutes and at 70 0C,
the microcentrifuge tubes were
incubated in a waterbath. After
doing so, the tubes were vortex-
mixed for 10 seconds. Next, the
tubes were placed on ice for 30
minutes.
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 To precipitate all cell debris,
the tubes were centrifuged at
14,000 rpm for 10 minutes. The
supernatant obtained was then
transferred to a new
microcentrifuge tube containing
isopropyl alcohol. Incubation at
room temperature was done for 10
minutes.
After incubation, the samples
were centrifuged at 12,000 rpm for
10 minutes. Finally, the DNA
pellets were washed with 70%
ethanol and then were allowed to
air dry. The pellet was then re-
suspended in 100 uL TE buffer.
B. DNA Extraction from Blood
The procedure for DNA
extraction from blood used the
Wang’s Method. But first, a whole
blood sample was first extracted.
The main reagent used for this
procedure is the Wang’s Lysis
solution which is composed of 1%
w/v Triton X-100, 0.32 M Sucrose,
5mM MgCl2 and 10 mM Tris-Cl at
pH 7.5.
There are other reagents aside
from the Wang’s Lysis Solution.
These reagents include the enzyme
reaction solution, sodium iodide
solution, Tris-EDTA buffer, 70%
ethanol, 40% absolute isopropanol
and 20 mg/mL Proteinase K stock
solution stored at -20 0C.
The enzyme reaction solution is
composed of 1% w/v SDS, 5 mM
EDTA and 10 mM Tris-Cl at pH
8.0.
The sodium iodide solution,
stored in the dark at room
temperature, is composed of 7.6 M
NaI, 20 mM EDTA and 40 mM
Tris-Cl at pH 8.0.
Lastly, the TE or Tris-EDTA
buffer was composed of 10 mM
Tris at pH 8.0 and 1 mM EDTA.
As for the procedure,
approximately 8 mL of blood was
first extracted. To the sample, 1
mg/mL EDTA-Na2 was added. The
blood solution was then divided
into 0.5 mL aliquots in
microcentrifuge tubes.
Wang’s lysis solution, in an
amount of 0.5 mL, was added next.
The resulting solution was then
centrifuged at 10, 000 x g for 20
seconds.
The supernatant was discarded,
1 mL of lysis solution was added to
the pellet and then the resulting
solution was mixed by inversion
for 30 seconds. Centrifugation at
10, 000 x g for another 20 seconds
was performed. The addition of 0.5
mL Wang’s lysis solution and
centrifugation was also repeated.
After centrifugation, the pellet
was re-suspended in 0.2 mL of
enzyme reaction solution.
0
Incubation at 37 C for 10 minutes
was performed next. Upon doing
so, 10 uL of Proteinase K solution
was added and incubated at 1 hour
for 37 0C, with occasional mixing.
0.3 mL of NaI solution was added,
and then gently inverted to mix. 0.5
mL of isopropanol was also added
and then gently inverted.
Centrifugation was once again
performed, at a rate of 10, 000 x g
for 10 minutes. Decantation was
used to remove the supernatant.
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 The pellet remained and to it, 1
mL of 40% isopropanol was added.
Centrifugation at 10, 000 x g was
performed for 5 minutes. The
supernatant was also removed.
The above step was performed
once again, but 70% ethanol was
used instead of 40% isopropanol.
Finally, the resulting pellet was
air-dried for 10 minutes and then
re-suspended in 0.1 mL TE buffer,
maintained at pH 8.0.
V. Results and Discussion
Out of these seven methods
discussed in the Introduction, the
method used for the DNA
extraction from bacteria is a
combination of the first and
second. To be more specific, it is
the Xanthogenate-SDS Method by
Tillett and Neilan, 2000.
DNA extraction starts with the
proper storage of the DNA sample.
Afterwards, disruption and lysis
begins.
Disruption and lysis can be
performed using a TER buffer. A
TER buffer is composed of Tris-
HCl, EDTA and RNAse A. Each
component has its function.
EDTA,ethylenetriamine
tetraacetic acid, is a known
chelating agent. It binds divalent
cations, specifically Magnesium
and Calcium which maintain the
cell membrane’s integrity. Upon
binding with the cations, the
membrane is disrupted and lysed.
Tris(hydroxymethyl)
aminomethane, or Tris, works as a
biological buffer. A buffer is of
tremendous importance in this
experiment because DNA is highly
pH-sensitive. The pKa of Tris is
equal to 8.1, which means that it
can prevent drastic pH shifts ad
maintain the pH at a range of 7.0 to
9.0. The second role of Tris is to
interact with the lipopolysaccharide
membrane and make it more
permeable in order for disruption
and then lysis to occur.
Since the membrane has been
disrupted and lysed, debris such as
RNA must be removed. RNAse A
is used to digest contaminating
RNA.
However, bacterial cells have
rigid cell walls that are rich in
polysaccharides. Consequently,
these are difficult to rupture
completely. Enzymes have been
used to rupture these cell walls but
the cells are not thoroughly lysed
and DNA yield is poor. Thus, the
XS buffer is added next.
The XS buffer is composed of
potassium ethyl xanthogenate, Tris-
HCl, sodium dodecyl sulphate and
ammonium acetate.
Potassium ethyl xanthogenate
is the salt of a xanthic acid. It is
formed by the reaction of
potassium hydroxide, ethanol and
carbon disulfide. In this
experiment, its main role is to
dissolve the cell wall of bacteria.
Upon dissolution, water-soluble
polysaccharide xanthates are
formed. Moreover, these xanthate-
forming compounds can inhibit
DNAse activity, by binding to
Magnesium and Calcium divalent
cations. Indeed, it serves to rupture
the cell wall and to destabilize the
membrane.
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 Another component of the XS
buffer is SDS or Sodium Dodecyl
Sulfate. Its main function is to act
as anionic surfactant and denature
the proteins that may act as
contaminants in the DNA sample.
Denaturation occurs because it is
amphiphillic and disrupts the non-
covalent bonds of proteins.
Ammonium acetate is also a
component of the XS buffer. It is
the salt of acetic acid and
ammonia. Approximately, it has a
pKa of 7.0. A salt, in this
experiment, functions to precipitate
the proteins out of the solution.
After the TER and XS buffer
have been added, the tubes were
incubated at 70 0C for an hour.
Incubation was done to ensure that
complete reaction took place.
At this point, it can be inferred
that RNA, protein and other
possible contaminants have been
removed from the “cellular soup”.
At the same time, the buffer served
to maintain the pH of the solution
at the physiological level.
To further separate the debris
from DNA, vortexing the DNA at
10 seconds was done. Care was
observed in vortexing since
genomic DNA is large and too
much agitation can shear the DNA.
In addition, the solution was
subject to an ice bath to aid in the
precipitation and then centrifuged
to finally separate the other cellular
components from DNA.
Since the genomic DNA is
heavier, it remained in the
supernatant. Afterwards, it was
treated to isopropanol. This was
done to precipitate the DNA, since
DNA is insoluble in polar
substances, such as alcohol.
Aggregation then occurs, and then
a white pellet is finally formed.
To remove excess alcohol and
those salts which are soluble in
alcohol, centrifugation was once
again performed. Washing in 70%
ethanol was done next, since
ethanol is volatile and easily
volatilizes itself from the DNA.
Finally, the pellet was
resuspended in TE buffer. A buffer
is needed, and not water, since
DNA hydrolyzes in water;
whereas, its pH is controlled in the
presence of a buffer.
The above mentioned protocol
is for bacterial DNA isolation.
Some of its steps are similar to that
of DNA isolation from human
tissues, specifically blood;
whereas, some are not. In this
paper, the method used for DNA
extraction from blood is that of
Wang’s.
DNA isolation from blood
starts with the proper storage of the
sample. For optimum conditions, it
must be stored at a temperature of
-20 0C to - 80 0C.
The first step, upon obtaining
the stored sample, is the addition of
an anticoagulant. This is needed so
as to avoid blood clotting and be
able to obtain whole blood. In the
market, the three main
anticoagulants used are EDTA-
Disodium, Heparin and Citrate.
Citrate gives poor DNA yields. The
mode of action of heparin consists
of inhibiting thrombin.
Consequently, upon inhibition,
fibrinogen is not converted to fibrin
and blood does not clot. However,
the problem concerning heparin is
7
that it interferes with downstream
applications of recovered DNA
(Beautler, et. al., 1990), though the
exact mechanism by which it
interferes has not been elucidated
yet.
For this reason, EDTA-
Disodium was chosen to be the
anticoagulant for this experiment.
The said reagent works by
chelating the calcium ions, which
render these inactive to participate
in coagulation. Moreover, EDTA-
Disodium does not interfere much
with recovered DNA downstream
applications.
A study (Ty, et. al, 2008)
shows that there is no significant
difference between the yield of
recovered DNA using EDTA-
Disodium and Heparin. Indeed,
choosing EDTA-Disodium over
Heparin is a matter of less
interference in downstream PCR
applications by the former reagent.
Figure 3. Average DNA yield
using three common
anticoagulants: EDTA, Citrate and
Heparin, respectively. (Ty, et. al.,
2008)
Upon addition of anticoagulant,
the blood was divided into aliquots
and Wang’s Lysis Solution was
added.
Wang’s Lysis Solution has
several components. These are
Triton X-100, sucrose, Magnesium
Chloride and Tris-Cl.
Before discussing the
components of the lysis solution, it
is important to note that
mammalian red blood cells or
erythrocytes do not contain nuclei.
Hence, genomic DNA cannot be
extracted from these. Another fact
is that in whole blood, there are
approximately 1000x more
erythrocytes than leukocytes which
contain nuclei. Thus, eliminating
erythrocytes is needed in order to
recover DNA that is high in both
yield and purity.
The elimination of erythrocytes
was done using the Wang’s Lysis
Solution. Triton X-100,
scientifically known as
octylphenolpoly(ethyleneglycoleth
er)x, is a component of the solution
and it functions as a non-ionic
detergent. Mainly, it solubilises the
proteins of the cell membranes,
rendering these easier to disrupt.
Sucrose is another component
of the lysis solution. It selectively
lyses erythrocytes by hypotonic
shock, since these are more
susceptible than leukocytes.
The next component is
Magnesium Chloride. It is a salt
and it functions to selectively
precipitate nuclei-containing cells
out of the solution. Also, the
Magnesium cations counteract the
Phosphate anions of the nucleic
acids and protects DNA from
degradation. In addition to this, the
buffer protects the solution from
pH shifts since the cell debris
generated can drastically alter it.
It can also be remarked that the
volume of the blood sample must
be equal to that of the lysis solution
8
so as to ensure complete lysis of
the erythrocytes.
Upon lysis, repeated
centrifugation was performed in
order to separate the debris from
the cellular pellet.
When the supernatant was
discarded and the pellet was
obtained, the enzyme reaction
solution was added. The enzyme
reaction solution contained SDS,
EDTA and Tris-Cl.
As discussed earlier, SDS
works as an anionic detergent that
disrupts the cell membrane, EDTA
chelates divalent cations and
disrupts cell integrity and Tris-Cl
works as a buffer. Incubation was
done so as to ensure complete lysis
of the cell.
Upon incubation, Proteinase K
was added. It is a protease with
broad specificity, has a high
activity and can digest proteins at
short times. Its optimum
temperature range is from 65 0C to
70 0C. Hence, the sample was
incubated at 70 0C to ensure
complete protein digestion.
A distinct characteristic of
Proteinase K that makes it suitable
for use is that it does not need
divalent cations, such as
Magnesium and Calcium. Hence,
EDTA can chelate these ions and
Proteinase K can still perform its
work of digesting proteins.
Since the proteins have already
been digested by the protease,
sodium iodide was added next.
Sodium iodide is a crystalline salt
that must be stored in the dark at
room temperature because it can
readily decompose. Furthermore,
low temperature can cause it to
recrystallize.
Biological compounds such as
polypeptides and proteins remain
soluble in high concentrations of
NaI; whereas, DNA are insoluble.
Thus, further addition of
isopropanol was able to selectively
precipitate DNA out of the
solution. Consequently, a white-
pellet was formed.
Centrifugation was once again
performed to separate the pellet
from the supernatant. The pellet
was treated with 40% isopropanol
to further remove alcohol-soluble
proteins and ensure that high purity
DNA is recovered.
Now that the DNA pellet has
been purified, 70% ethanol was
added to it. Air-drying was
performed next. Ethanol easily
volatilizes; thus, what remains in
the solution is DNA.
Lastly, DNA was re-suspended
in TE buffer. TE buffer, not water,
was used since DNA hydrolyzes in
water, whereas, the buffer protects
it from pH shifts.
The recovered DNA can be
used for several purposes. It can be
applied in PCR, restriction enzyme
digestion, molecular cloning and
DNA sequencing.
After agarose gel
electrophoresis in 1x TAE was
performed on the recovered DNA,
it was viewed under UV light. The
resulting electrophoresis is as
follows:
9

Figure 4. Electrophoresis of
Genomic DNA Purified from
Bacteria (Lanes 1-4) and Blood
(Lanes 7-9). Lanes 5, 6 and 10
served as markers.
In Lane 1, no distinct band can
be seen. Mishandling of the DNA
might have occurred in the process
of disposing the supernatant. The
pellet, which might have been too
small to settle at the bottom of the
microcentrifuge tube, might have
been disposed of. Thus,
supernatants must be kept until the
analysis of DNA has been
completed since it might contain
valuable DNA.
In Lanes 2 to 4, the distinct
bands show the high-molecular
weight of bacterial genomic DNA.
The faded bands at the bottom
show cellular debris, particularly
RNA, which was not degraded by
RNAse A since the enzyme was
not available in the laboratory.
Lanes 5 and 6 contained the
blood marker. No observable band
was seen; hence, no DNA was
extracted. The cause of this
problem can be attributed to the
storage of DNA. Earlier, it was
discussed that blood samples for
DNA isolation must be kept at a
temperature range of -20 0C to - 80
0
C. Such range was not available
and DNA became destabilized.
Lanes 7 to 9 should
theoretically contain blood from
genomic DNA. Such was not the
case. Several mistakes can cause
this. The blood sample might have
not been properly stored, the lysis
solution might have not been
enough to lyse the erythrocytes or
the small pellet might have been
disposed of.
Lastly, Lane 10 served as the
marker for the experiment.
Another figure shows the
analysis using electrophoresis, this
time using a kit for bacterial
genomic DNA extraction.
Figure 5. Electrophoresis of
Purified Genomic DNA of Bacteria
from kit (Lanes 1 and 2), Bacteria
from Standard Protocol (Lanes 3
and 4) and blood (Lanes 7 and 8).
Lanes 5, 6 and 9.
As can be seen in this figure,
using DNA kits does not ensure a
no-fail experiment. No DNA bands
can be observed from that of Lanes
1 and 2 since the water bath was
not maintained at 70 0C and lysis
might have not occurred. Indeed,
only proper handling of the
10
 experiment protocol can ensue that
DNA is recovered.
VI. Conclusion and
Recommendations
At the end of the experiment, it
can be concluded that bacterial DNA
was successfully extracted using the
Tillett and Neilan Method.
This is supported by the fact
that distinct bands were seen in the gel
electrophoresis.
Indeed, the protocol used was
efficient, fast, relatively cheap and
safe, when compared to organic
extraction methods.
A kit was also employed in this
experiment. However, a kit does not
always guarantee a better result than
manual techniques since the proper
handling of the samples and reagents is
still the key to a successful experiment.
For genomic DNA extraction
from whole blood, the Wang’s Lysis
Method was used.
As can be seen in the gel
electrophoresis, no bands were
observed due to both the improper
storage of the samples and practice of
laboratory techniques.
The recovered DNA from these
experiments has several applications.
These include PCR, Southern Blotting,
molecular cloning and DNA
sequencing, to name a few.
VII. References
1. Tillett, D. and B. A. Neillan.
Xanthogenate Acid Isolation from
Cultured and Environmental
Cyanobacteria. Journal of
Phycology, 2000.
2. Wang, et. al., Purification of
Genomic DNA from Human
Whole Blood by Isopropanol-
Fractionation with Concentrated
NaI and SDS. Japan: Osaka
Research Laboratories, 1994.
3. Khosravinia, et. al. Influence of
EDTA and Magnesium on DNA
Extraction from Blood Samples
and Specificity of Polymerase
Chain Reaction. India: Lorestan
University, 2006.
4. Ty, et. al., Isolation of DNA
from Blood Samples and Body
Fluids Using E-Z 96 Mag-Bind
Blood System. VWR International,
Issue 21, 2008.
5. Beutler, et. al. Interference of
Heparin with the Polymerase Chain
Reaction. Journal of
Biotechnology, 1990.
I hereby certify that I have given
substantial contribution to this
report.
Lopez, Richard
Palmario, Katrina Marijyke
Tan, Elaine
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