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ESTIMATION OF ANTIOXIDANT

ENZYME(CATALASE): A BIOMARKER
FOR TYPE 2 DIABETES
DEPARTMENT OF BIOTECHNOLOGY
INTERNAL GUIDE
Ch.N.VANI

EXTERNAL GUIDE
DR. KAISER JAMIL

SHAYISTA NAAZ
09J21A2316

AIM AND OBJECTIVES


Aim is to estimate the effect of antioxidant (catalase)

activity in Type2 Diabetes.


To identify and isolate human genomic DNA and

amplify the antioxidant (catalase) gene using specific


primers for catalase gene with polymerase chain
reaction.
To use antioxidant (catalase) as a potential biomarker
for type 2 diabetes.

INTRODUCTION
DIABETES:
Type I diabetes:
In type 1 diabetes, the pancreas
undergoes an autoimmune attack
by the body itself, and is rendered
incapable of making insulin.
Type II Diabetes:
Insulin resistance
When sugar cannot enter the cells
they start building up in the blood
at high amounts in the blood
stream is called hyperglycemia.

ROLE OF ANTIOXIDANT IN DIABETES:


Anti-oxidants are involved to reduce diabetic complications

because they are involved to fight or destroy free radicals.


These free radicals in hyperglycemia by glucose toxicity, glucose
auto oxidation and oxidative phosphorylation produces ROS and
releases free radicals and these are involved in oxidative stress
and leads to destroy the pancreas.
The beta cells are inhibited by their function and destroyed by
free radicals.
The above process will prone to diabetic complications so the
antioxidants role here is to inhibit activity of free radicals and
avoiding diabetic long-term complications.

MECHANISM OF ACTION CATALASE


Catalase is a common enzyme found in nearly all living

organisms that are exposed to oxygen, where it functions to


catalyze the decomposition of hydrogen peroxide to water
and oxygen.
Catalase has one of the highest turnover numbers of all
enzymes; one molecule of catalase can convert millions of
molecules of hydrogen peroxide to water and oxygen per
second.

catalase
2H2O2

2H2O + O2

Function of CAT gene


This gene encodes catalase, a key antioxidant enzyme in the

bodies defense against oxidative stress.


Catalase is a heme enzyme that is present in the peroxisome of
nearly all aerobic cells.
Cytogenetic Location : 11p13
The CAT gene is located on the short (p) arm of chromosome 11 at

position 13. It contains 13 exons


CAT Gene located 11p13

MATERIALS AND METHOD


Biochemical analysis of antioxidant (catalase)

Estimation of antioxidant enzyme


Molecular analysis of antioxidant (catalase)

Isolation of genomic DNA


Quantification of DNA
Amplification of DNA

ESTIMATION OF PROTEIN
1 ml of

ESTIMATION OF CATALASE

GENOMIC DNA ISOLATION FROM BLOOD


300

Supernatant was transferred carefully to 680

QUNTIFICATION BY SPECTROPHOTOMETER
The isolated DNA is of pure quality and is not contaminated

with proteins, RNA and other contaminants, since A260/A280 lies


between 1.8 to 2.0. Which shows DNA is of pure form.

Polymerase chain reaction(PCR):


The DNA obtained were used as templates for PCR using

specific primers.
Reagents

Volume

Water

18.3l

Buffer

2.5l

dNTPs

1l

Forward Primers

1l (20 pmol/l)

Reverse Primers

1l (20 pmol/l)

Taq Polymerase

0.2l

DNA sample

1l

The reagents as mentioned in the proportions were added

to the nuclease free PCR tubes and placed them in a


cycler and the following PCR conditions were
maintained:
Stage 1: 94oC 5 min
Stage 2: 35 cycles
Step 1: 95oC 1 min
Step 2: 60oC 1 min
Step 3: 72oC 1 min
Stage 3:
Step 1: 72oC 5 min
Step 2: 15oC

CONCLUSION

THANK YOU

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