MATERIALS AND METHOD

BLOOD SAMPLE COLLECTION

Blood samples were collected after obtaining the patients consent and ethical
clearance from the Ethical Committee by Jawaharlal Nehru Institute of Advanced Studies
(JNIAS). Blood samples were collected in vials containing anticoagulant EDTA
vacuumed tubes and kept at -20oC within 30 minutes after removal from patients from
Mahaveer hospital, Hyderabad and brought to JNIAS in cool box containing ice.

BIOCHEMICAL ANALYSIS OF CATALASE:

Serum separation for protein estimation and catalase activity:
SERUM:
It is most commonly used by clinical reference laboratories. Serum is nothing
but plasma, after excluding fibrinogen and other blood clotting factors. Fibrinogen is a
type of protein, responsible for coagulation of blood, by converting itself into fibrin.
Whereas, plasma is defined as the medium of blood, in which blood cells (red blood cells
and white blood cells) and other components are suspended.

Components of serum:
Serum is made up of non-clotting proteins, glucose, nutrients, electrolytes,
hormones, antigens, antibodies and other particles. While the components of plasma are
same as that of serum, except for fibrinogens and clotting factors that are absent in serum.
In short, the relation between serum and plasma can be put as, 'serum = plasma after
removal of clotting factors'.

Percentage volume of serum:

Blood plasma accounts to more than half of the volume of blood. Out of this 90%
of the volume is made up of water; the remaining 10% comprises the blood cells,
nutrients and other parts. Since plasma accounts to about 55 percent of blood, definitely

the percentage volume of serum is lesser than this. After all, serum is plasma devoid of
clotting

factors.

Isolation procedure:
Separation of serum is more tedious and time-consuming than plasma extraction.
For isolation of serum, first a blood sample is allowed to clot, after which the coagulated
blood is centrifuged. The liquid supernatant formed at the top portion is serum. The
procedure for extraction of plasma is very simple; blood sample is spun by using a
centrifuge apparatus. The heavier blood cells settle at the bottom, and blood plasma is
collected from the upper layer.
Collection of blood: 2-3 ml of blood was collected by venepuncture in
plastic tubes without any anticoagulant. The tubes were centrifuged for
10 minutes at 4000 rpm. Supernatant serum was pipetted out with a
micropipette, transferred to an Eppendorf tube, and stored in deep
freeze. Biochemical estimation was performed from these sera in the
following methods.

Usage of serum in medicine:

Serum is used for regular blood group testing (or checking blood types) and
disease diagnostic purposes. On the other hand, blood plasma is delivered to supplement
lack of blood components in patients. It can also be used for detection of diseases. Before
storage and after separation, plasma is treated with an anticoagulant substance to prevent
clotting.
It is expected that the composition of the serum proteome can provide valuable
information about the state of the human body in health and disease and that this
information can be extracted via quantitative proteomic measurements. Hence, we have
quantified protein in human by using spectrophotometer.

PROTEIN- SERUM ANALYSIS:

Serum is isolated from blood samples of both normal and diabetic patients and performed protein
analysis to evaluate the usefulness of serum total protein.

ESTIMATION OF PROTEIN:
We have estimated the protein using the Lowry method. The Lowry assay-Protein by
Folin Reaction (Lowry et al., 1951) has been the most widely used method to estimate
the amount of protein in biological samples.

PRINCIPLE:
The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein
will produce a blue purple color complex , with maximum absorption in the region of 595
nm wavelength, with Folin- Ciocalteau reagent which consists of sodium tungstate
molybdate and phosphate. Thus the intensity of color depends on the amount of these
aromatic amino acids present and will thus vary for dierent proteins. Estimation
techniques use Bovin Serum Albumin (BSA) universally as a standard protein.

REAGENT REQUIRED:
1.

BSA stock solution (1mg/ml)

2.

Sodium Carbonate anhydrous

3.

Sodium hydroxide

4.

Copper sulphate

5.

Sodium potassium tartarate

6.

F.C reagent (Phenol reagent)

Materials required:
1.

Test tubes

2.

Pipettes

3.

Beakers and flasks

4.

Weighing balance

5.

Cuvettes

6.

Spectrophotometer

REAGENTS PREPARED:
Lowry

A: 2%

Sodium

Carbonate

anhydrous

in

0.1M

Sodium

hydroxide. (0.56g NaOH+2.86g Na2Co3 in 100 ml water).

Lowry B: 1% CuSo4 in distilled water ( 0.28g of CusO4 in 20ml
distilled water).
Lowry C: Sodium potassium tartarate( 0.56g in 20ml of distilled
water).
Lowry stock reagent: 49ml of Lowry A + 0.5ml of Lowry B+ 0.5ml
of Lowry C
F.C reagent: Phenol reagent (2N) was diluted in water in 1:1 ratio.
To estimate the amount of protein in an unknown sample, we should first prepare
a standard graph using a known protein sample. We have used Bovine serum albumin
(BSA) as known sample to obtain the standard graph.

PROCEDURE FOR THE PREPARATION OF STANDARD

GRAPH:
We have prepared different dilutions of BSA solutions by mixing stock BSA
solution (1 mg/ ml) water and F.C reagent in the test tube. The nal volume in each of the
test tubes is 2 ml.
BSA(l)
Water(l)
FC

0
1.8
200

200
1.6
200

FOR

THE

400
1.4
200

600
1.2
200

800
1
200

1000
0.8
200

reagent(l)

PROCEDURE

ESTIMATION

SAMPLE USING THE STANDARD GRAPH:

OF

UNKNOWN

The serum was separated from the normal and diabetes patients blood sample.
1ml of lowry stock reagent was taken in test tube. 10 micro liters of serum sample was
added to the reagent which was present in the test tube. The test tubes were kept for
incubation for about 30mins at room temperature.
After incubation 100 micro liters of FC reagent was added. The test tube were kept
for incubation at room temperature for another 30mins. After incubation, 1ml of the
mixture was taken in a cuvette to read the OD value using spectrophometer. 595nm.
Wavelength was used. The above steps were repeated for all samples.

ESTIMATION OF CATALASE:
Chemicals required:
1. Potassium di-hydrogen orthophosphate
2. Di-potassium hydrogen phosphate
3. Hydrogen peroxide solution

Materials required:
1.
2.
3.
4.

Beakers
Pipettes
Cuvettes
Spectrophotometer

Reagents prepared:
Phosphate buffer: Potassium di-hydrogen orthophosphate was mixed
with di-potassium hydrogen phosphate with pH maintained at 7.
Hydrogen peroxide solution: 30% H2O2 was diluted 10 times in
water (1 ml of H2O2 in 9ml water). This diluted solution is again diluted 3
times (1ml of diluted H2O2 in 2ml of water) bringing it to 1% solution
(30mM).
Catalase stock solution: 840 micro liter of H2O2 solution was added
in 299.160ml phosphate buer and a stock of 300ml was prepared.

PROCEDURE:

790micro liter of water was taken in a cuvette.200 microliter of

reagent was added to it. 10 microliter of serum sample was added.
Adjusted the wavelength to 240nm and noted down the OD values
using the time scan measurement in the spectrophotometer.
Using the obtained OD values of protein and catalase we found out the activity of the
catalase enzyme by substituting in the formula below:
Catalase activity= Final OD/extinction coefficient*volume of sample*protein
concentration

MOLECULAR ANALYSIS OF CATALASE

ISOLATION OF GENOMIC DNA FROM HUMAN BLOOD SAMPLE
DNA was isolated from the blood samples by a rapid non-enzymatic method by salting
out cellular proteins with saturated solution and precipitation by dehydration (Alluri et
al., 2005).

MATERIALS REQUIRED:
1.
2.
3.
4.

5.

Autoclaved eppendor
Autoclaved micropipettes
Autoclaved micro tips
Autoclaved distilled water
Eppendor stand

PREPARATION OF REAGENTS
The reagents were prepared as described below:

RBC LYSIS BUFFER/ TKM 1

Chemicals

(100ml)

(50ml)

Tris-HCL (10mM)

0.121

0.061

EDTA ( 2mM)

0.0744

0.0372

KCl

(10mM)

0.0745

0.03725

MgCl2 (10mM)

0.2033

0.10165

Tris is first dissolved in few ml of autoclaved distilled water and the pH is adjusted to 7.6.
By using 0.1% NaOH Then EDTA is dissolved followed by other chemicals.

Cell Lysis Buffer/ TKM2 (100ml) and (50ml)

Chemicals

(100ml)

(50ml)

Tris-HCL (10mM)

0.121

0.061

EDTA ( 2mM)

0.0744

0.0372

KCl

(10mM)

0.0745

0.03725

MgCl2 (10mM)

0.2033

0.10165

NaCl (0.4M)

2.3376

1.1688

Tris is first dissolved in few ml of autoclaved distilled water and the pH is adjusted 7.6
with NaOH. Then EDTA is dissolved followed by other chemicals and the volume is
made up to 100 ml with distilled water.
10% SDS (10 ml): 1gm of SDS was dissolved in 10 ml of autoclaved distilled water.
0.6M NaCl: 0.8765 g of NaCl was dissolved in 25 ml of distilled water.
TE Buffer
Chemical

Amount

Tris hydrogen chloride (HCl) (10mM) pH 8

Ethylene diamine tetra acetic acid (EDTA) (1mm)

0.030 g
0.009 g

Tris is dissolved in few ml of autoclaved distilled water, after adjusting the pH, EDTA is
dissolved, and the volume is made up to 25 ml.
70 % Ethanol Dissolve 7 ml of absolute Ethanol in 10 ml of distilled water.

PRINCIPLE:
RBC Lysis Buffer and Triton X 100 is used to remove the RBCs:Since RBC has no charge on their plasma membrane, non- ionic detergent called, Triton
X 100 removes them out. KCl and MgCl 2 in TKM1 helps in lysis of the RBC cell
membrane and EDTA acts as a divalent ion chelator (it contains di-sodium atom). Hence,
it helps in de-activating the metallozymes as DNAses. Tris acts as a buffering agent
maintaining the pH at 7.6 for the proper function of the lysis buffer. In addition, it helps
in solubility of the ions so that they do not precipitate out.
Centrifugation at 10000 rpm for 5 minutes, after incubation with RBC lysis buffer
step separates out the lysed RBCs in the supernatant and intact lymphocytes precipitate
out as pale colored pellet.
TKM2 and 10% SDS are used to lyse the lymphocytes:TKM2 or Cell lysis buffer has a higher concentration of MgCl 2, KCl and NaCl to
lyse both the cell and the nuclear membrane. KCl also acts as solubilizer of proteins.
NaCl acts as extractor of RNA and used in salting out of proteins .SDS acts as anionic
detergent and both acts on anionic lymphocytic cell membranes and help in their lysis
deactivate the negatively charged proteins.
6M NaCl is added to precipitate the proteins by salting out method. Centrifugation at
10000 rpm for 5 mins helps in the precipitation of the aggregates of the proteins and cell
debris as pellet and the supernatant contains the DNA strands in a solubilized form.
Addition of the supernatant to cold absolute ethanol dehydrates the DNA and
extracted as visible strands. The visible DNA threads are precipitated as pellet, washed

using 70% alcohol, and again precipitated to remove agents like MgCl 2, EDTA, KCl,
NaCl that can inhibit Taq Polymerase during PCR of these samples.

PROCEDURE:
300 l of blood sample was taken in eppendorff. 800 l of TKM1 and 1 drop of
100% Triton X 100 was added to it, mixed well, and incubated for 5 minutes. Centrifuged
at 10000 rpm for 5 minutes, and then supernatant was discarded. To the pellet 800 l of
TKM1 was added and steps 2 and 3 are repeated until a white pellet is obtained. To the
pale pellet, 300 l of TKM2 and 80 l of 10% SDS was added and incubated for 30
minutes. 80 l of 6M NaCl was add and mixed well by tapping for 5 minutes.
Centrifuged at 10000 rpm for 5 minutes. Supernatant was transferred carefully to 680 l
of cold absolute Ethanol. Centrifuged at 10000 rpm for 5 minutes. Supernatant was
discarded and 300 l of 70% absolute Ethanol was added to the DNA pellet.Centrifuged
at 10000 rpm for 5 minutes and the pellet was air dried. To the dried pellet, 50 l of TE
buffer was added for hydration of DNA and preserve at freezing temperature. Detection
of DNA in the Isolated Samples Using 0.8% of Agarose Gel by Electrophoresis.

ISOLATION OF GENOMIC DNA FROM HUMAN BLOOD

SAMPLE BY SSC PROTOCOL
REAGENTS REQUIRED:
1. 1XSSC buffer
2. Sodium acetate
3. Proteinase K
4. Phenol chloroform isoamyl alcohol
5. Cold 100% ethanol
6. 10:1 TE buffer

7. EDTA vacationer tubes

PRINCIPLE:
Phenol chloroform isoamyl alcohol is used to extract proteins and lipids away from
DNA. 0.2M sodium acetate and 2M sodium acetate precipitates the protein in the blood.
Proteinase K, it is a protein digesting enzyme, it digest the proteins in DNA solution.
TE buffer (10:1) which maintain PH and it protects the DNA from degradation. Ethanol
(80%) precipitates the DNA. SSC buffer increases ionic strength, so precipitation of DNA
or RNA is increases

PROCEDURE:
Blood samples typically were obtained as 500 l of whole blood stored in EDTA
vacationer tubes frozen at -70deg C. Thaw the frozen samples, and to each 500 l
sample, 400 l 1X SSC buffer were added, and mixed well. Centrifuged for 1 minute at
12,000 rpm in a microcentrifuge. 1 ml of the supernatant was removed and discard into
disinfectant. 500 l of 1X SSC buffer was added, vortex, centrifuged as above for 1
minute, and removed all of the supernatant.187 l of 0.2M NaOAc added to each pellet
and vortex briefly. Then 12.5 l of 10% SDS added and 2.5 l of proteinase K (20 mg/ml
H2O), vortex briefly and incubate for 1 hour at 55degC.
60 l phenol/chloroform/isoamyl alcohol was added and vortex for 30 seconds.
Centrifuged the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube. Carefully
removed the aqueous layer to a new 1.5 ml microcentrifuge tube, 500 l of cold 100%
ethanol was added, mixed and incubate for 15 minutes at -20deg C. Centrifuged for 2
minutes at 12,000 rpm in a microcentrifuge. the supernatant was decant and drain. 90 l
TE buffer was added, vortex and incubate at 55degC for 10 minutes. 10 l 2M sodium
acetate was added and mixed. Add 250 l of cold 100% ethanol, mixed, and centrifuged
for 1 minute at 12,000 rpm in a microcentrifuge. the supernatant was decant and rinsed
the pellet with 500 l of 80% ethanol. Centrifuged for 1 minute at 12,000 rpm in a
microcentrifuge. the supernatant was decant, and dried
Resuspended the pellet by adding 35 l of 10:1 TE buffer.

the pellet (or until dry).

AGAROSE GEL BY ELECTROPHORESIS

MATERIALS REQUIRED:
1.
2.
3.
4.
5.

Horizontal electrophoresis unit

Gel plate
Combs
Adhesive tapes
10T micropipette and autoclaved tips

REAGENT PREPARATION:
10 x TAE Buffer (100) ml:

Solution A: 19.36g of Tris was dissolved in 50 ml of distilled water.

Solution B: 1.86g of EDTA was dissolved in 10ml of distilled water.

Solution C: 8 ml of B solution was added to solution A and 4.36ml of acetic acid was
added. Then the volume was made up to 100ml with distilled water.
1X TAE Buffer: 30ml of 10X TAE Buffer was dissolved in 270 ml of distilled water to
make 1:10 dilution.
0.8% Agarose: 0.2g of Agarose was dissolved in 25ml of 1X TAE Buffer.
1% Ethidium bromide solution: 0.1g of Ethidium bromide was dissolved in 10ml
distilled water. Gel loading solution and dye used was 6X concentrated and was
obtained readymade.

PRINCIPLE:
Electrophoresis: An electric field is developed across the Agarose gel with
incorporated DNA samples. The DNA being negatively charged migrates towards anode.
The Ethidium bromide acts as an interchelating agent and incorporates itself into the
DNA strands. Since Ethidium bromide fluoresce under UV rays, hence site of
fluorescence in the gel detects presence of DNA.
Bromophenol blue is used as loading dye to track the movement of the sample. It
is mixed well with the sample. In addition, it increases the density of the mixture, so that
they reside down at the bottom of the well and are diffused in the gel.

Gel electrophoresis: Gel documentation:

Fig: Agarose Gel Electrophoresis

Fig: Gel Documentation System

PROCEDURE:
Open sides of the gel plate was closed using adhesive taps. Combs was placed,
7 l of Ethidium bromide solution was added to the cold molten Agarose and poured it in

the gel plate. Keeps it resting for casting of the gel for 15 20 minutes. The taps and
comb were removed carefully. 1X TAE Buffer was poured in the unit tank and the gel
was placed by placing the wells at cathode end. 3 l of the loading concentrate was mixed
with 4 l of the DNA sample on a piece of parafilm. 7 l of the mixture was added into
the well. Wires were connected and the volts were set at 60. Gel was runned at 60V for
20 30 minutes. After 20-30 minutes gel was observed under UV in a transiliuminator.

SPECTROPHOTOMETRY:
In chemistry, spectrophotometer is the quantitative measurement of the reflection or
transmission properties of a material as a function of wavelength. It is more specific than
the general term

electromagnetic spectroscopy in that spectrophotometer deals

with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved
spectroscopic techniques.
Spectrophotometer involves the use of a spectrophotometer. A spectrophotometer is
a photometer that can measure intensity as a function of the light source wavelength.
Important features of spectrophotometers are spectral bandwidth and linear range of
absorption or reflectance measurement.

Fig: Spectrophotometry
Because DNA and RNA absorb ultraviolet light, with a absorption peak at 260nm
wavelength, spectrophotometers are commonly used to determine the concentration of
DNA in a solution. Inside a spectrophotometer, a sample is exposed to ultraviolet light at
260 nm, and a photo-detector measures the light that passes through the sample. The
more light absorbed by the sample, the higher the nucleic acid concentration in the
sample.
Using the Beer-Lambert law it is possible to relate the amount of light absorbed to the
concentration of the absorbing molecule. At a wavelength of 260 nm, the extinction
coefficient for double-stranded DNA is 50 (g/ml)-1 cm-1; for single-stranded DNA and
RNA it is 38 (g/ml)-1 cm-1. Thus, an optical density (or OD) of 1 corresponds to 50
g/ml for double-stranded DNA, 38 g/ml for single-stranded DNA and RNA. This
method of calculation is valid for up to an OD of at least 2.

Fig: A spectrophotometer measures how much light of a certain wavelength is

absorbed by a liquid

DNA concentration (g/ml) = OD260 x 100 (dilution factor) x 50 g/ml

1000

PROCEDURE:
99 l of TE buffer was taken in a cuvette and calibrated the spectrophotometer at 260nm
as well as 280nm. 1 l of each DNA sample to 99 l TE (Tris-EDTA buffer) and mixed
well, 2.9 ml of water was added to the cuvette.
TE buffer and water used as a blank in the other cuvette of the spectrophotometer.
The OD260 and OD280 values on spectrophotometer were noted.

POLYMERASE CHAIN REACTION

PRINCIPLE:
Polymerase chain reaction invitro was designed first by Karry Mullis in 1983. It
follows the process of DNA replication using temperature variations with a help of a
thermocycler. This process include five major steps , at specific accurate temperature for
each step for exact specificity of the amplification or duplication of the specific DNA
sequence or gene out of the whole genomic DNA sequence. This is possible by using
specific complementary forward and reverse primers that specified the region of
duplication. The enzyme used for the amplification is generally consists of 3 end to 5
end extension and 5 end to 3 end exonuclease activity. The enzyme used is called Taq
polymerase, which is extracted from thermostable bacteria Thermus aquaticus, generally
found in hot springs.
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of
copies of a gene. This is necessary to have enough starting template for sequencing.

The cycling reactions:

There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This
is done on an automated cycler, which can heat and cool the tubes with the reaction
mixture in a very short time.

1. Denaturation at 94C :
During the denaturation, the double strand melts open to single stranded DNA, all
enzymatic reactions stop (for example : the extension from a previous cycle).

2. Annealing at 54C :
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are
constantly formed and broken between the single stranded primer and the single stranded
template. The more stable bonds last a little bit longer (primers that fit exactly) and on
that little piece of double stranded DNA (template and primer), the polymerase can attach
and starts copying the template. Once there are a few bases built in, the ionic bond is so
strong between the template and the primer, that it does not break anymore.

3. Extension at 72C :
This is the ideal working temperature for the polymerase. The primers, where
there are a few bases built in, already have a stronger ionic attraction to the template than
the forces breaking these attractions. Primers that are on positions with no exact match,
get loose again (because of the higher temperature) and don't give an extension of the
fragment. The bases (complementary to the template) are coupled to the primer on the 3'
side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,
bases are added complementary to the template).

Fig: Polymerase Chain Reaction

Fig: The different steps in PCR

Because both strands are copied during PCR, there is an exponential increase of the
number of copies of the gene. Suppose there is only one copy of the wanted gene before
the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4
copies, three cycles will result in 8 copies and so on.

Fig: The exponential amplification of the gene in PCR.

Procedure for making 25l of PCR reaction:
Reagents

Volume

Water

18.3l

Buffer

2.5l

dNTPs

1l

Forward Primers
Reverse Primers
Taq Polymerase
DNA sample

1l (20 pmol/l)
1l (20 pmol/l)
0.2l
1l

PROCEDURE:
Content was mixed well gently by tapping. The amplification was carried out in a thermo
cycler for 30-35 cycles. After amplification, amplified samples were analyzed using
agarose gel electrophoresis. Store the amplified samples were stored at freezing
temperature for further analysis.

AGAROSE GEL BY ELECTROPHORESIS

:MATERIALS REQUIRED
1.
2.
3.
4.
5.

Horizontal electrophoresis unit

Gel plate
Combs
Adhesive tapes
10T micropipette and autoclaved tips

REAGENT PREPARATION:
10 x TAE Buffer (100) ml:

Solution A: 19.36g of Tris was dissolved in 50 ml of distilled water.

Solution B: 1.86g of EDTA was dissolved in 10ml of distilled water.

Solution C: 8 ml of B solution was added to solution A and 4.36ml of acetic acid was
added. Then the volume was made up to 100ml with distilled water.
1X TAE Buffer: 30ml of 10X TAE Buffer was dissolved in 270 ml of distilled water to
make 1:10 dilution.
1.5% Agarose: 0.75g of Agarose was dissolved in 50ml of 1X TAE Buffer.
1% Ethidium bromide solution: 0.1g of Ethidium bromide was dissolved in 10ml distilled
water. Gel loading solution and dye used was 6X concentrated and was obtained
readymade.

Gel electrophoresis: Gel documentation:

Fig: Agarose Gel Electrophoresis

Fig: Gel Documentation System

PROCEDURE:
Open sides of the gel plate was closed using adhesive taps. Combs was placed,
7l of Ethidium bromide solution was added to the cold molten Agarose and poured it in

the gel plate. Keeps it resting for casting of the gel for 15 20 minutes. The taps and
comb were removed carefully. 1X TAE Buffer was poured in the unit tank and the gel
was placed by placing the wells at cathode end. 3 l of the loading concentrate was mixed
with 4 l of the DNA sample on a piece of parafilm. 7 l of the mixture was added into
the well. Wires were connected and the volts were set at 60. Gel was runned at 60V for
20 30 minutes. After 20-30 minutes gel was observed under UV in a transiliuminator.