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 di erent proteins. Estimationff

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 final volume in each of the

test tubes is 2 ml.

BSA(μl) 0 200 400 600 800 1000

Water(μl) 1.8 1.6 1.4 1.2 1 0.8

FC reagent(μl) 200 200 200 200 200 200

 

PROCEDURE FOR THE ESTIMATION OF UNKNOWN SAMPLE

USING THE STANDARD GRAPH:

The serum was separated from the normal and diabetes patient’s 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. Beakers

2. Pipettes

3. Cuvettes

4. 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 buffer 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. Autoclaved eppendorff

2. Autoclaved micropipettes

3. Autoclaved micro tips

4. Autoclaved distilled water

5. Eppendorff 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 0.030 g

Ethylene diamine tetra acetic acid (EDTA) (1mm) 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 RBC’s:-

Since RBC has no charge on their plasma membrane, non- ionic detergent called, Triton

X 100 removes them out. KCl and MgCl2 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 MgCl2, 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 MgCl2, EDTA, KCl,

NaCl that can inhibit Taq Polymerase during PCR of these samples.

PROCEDURE:

300 μl of blood sample was taken in eppendorff. 800 μl 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 μl of

TKM1 was added and steps 2 and 3 are repeated until a white pellet is obtained. To the

pale pellet, 300 μl of TKM2 and 80 μl of 10% SDS was added and incubated for 30

minutes. 80 μl 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 μl

of cold absolute Ethanol. Centrifuged at 10000 rpm for 5 minutes. Supernatant was

discarded and 300 μl 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 μl 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 the pellet (or until dry).

Resuspended the pellet by adding 35 l of 10:1 TE buffer.

AGAROSE GEL BY ELECTROPHORESIS

MATERIALS REQUIRED:

1. Horizontal electrophoresis unit

2. Gel plate

3. Combs

4. Adhesive tapes

5. 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.

Fig: Agarose Gel Electrophoresis Fig: Gel Documentation System

PROCEDURE:

Open sides of the gel plate was closed using adhesive taps. Combs was placed,

7 μl 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

Electrical field

Positive electrodeAgarose gelElectrophoresis

chamber

Gel electrophoresis:

A compact system for superior results

Gel documentation:

was placed by placing the wells at cathode end. 3 μl of the loading concentrate was mixed

with 4 μl of the DNA sample on a piece of parafilm. 7 μl 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 (μg/ml)-1 cm-1; for single-stranded DNA and

RNA it is 38 (μg/ml)-1 cm-1. Thus, an optical density (or “OD”) of 1 corresponds to 50

µg/ml for double-stranded DNA, 38 µg/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 μl 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 94°C :

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 54°C :

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 72°C :

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 25µl of PCR reaction:

Reagents Volume

Water 18.3µl

Buffer 2.5µl

dNTPs 1µl

Forward Primers 1µl (20 pmol/µl)

Reverse Primers 1µl (20 pmol/µl)

Taq Polymerase 0.2µl

DNA sample 1µl

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. Horizontal electrophoresis unit

2. Gel plate

3. Combs

4. Adhesive tapes

5. 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.

Electrical field

Positive electrodeAgarose gelElectrophore

sis chamber

Gel electrophoresis:

A compact system for superior results

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,

7μl 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 μl of the loading concentrate was mixed

with 4 μl of the DNA sample on a piece of parafilm. 7 μl 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.