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ESTIMATION OF ANTIOXIDANT ENZYME(CATALASE): A BIOMARKER
FOR TYPE 2 DIABETES
DEPARTMENT OF BIOTECHNOLOGY
INTERNAL GUIDE EXTERNAL GUIDECh.N.VANI DR. KAISER JAMIL
SHAYISTA NAAZ09J21A2316
AIM AND OBJECTIVESAim 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.
INTRODUCTIONDIABETES: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 CATALASECatalase 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 geneThis 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 : 11p13The 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 METHODBiochemical 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 PROTEIN1 ml of lowry stock +10 μl of serum sample + incubation for 30
min
100 μl of F.C reagent + incubation for 30 min
Analyzing using UV-VIS spectrophotometer at 595 nm
ESTIMATION OF CATALASE790 μl of water+200 μl of reagent+ 10 μl of serum
Analyze using UV-VIS spectrophotometer at 240nm
Calculate the protein content
300 μl of blood sample + 800 μl of TKM1 and 1 drop of 100% Triton X 100 +incubate d for 5 min
Centrifuged at 10000 rpm for 5 minutes + then supernatant was discarded.
To the pellet 800 μl of TKM1 repeated for 2-3 times till white pale pellet is formed
To the pellet 800 μl of TKM1 repeated for 2-3 times till white pale pellet is formed
300 μl of TKM2 and 80 μl of 10% SDS + incubation for 30 min
80 μl of 6M NaCl + tapped for 5 min + centrifuged at 10000 rpm for 5min
GENOMIC DNA ISOLATION FROM BLOOD
Supernatant was transferred carefully to 680 μl of cold absolute ethanol + Centrifuged at 10,000 rpm for 5 minutes + discard
supernatant.
300 μl of 70% absolute ethanol was added to the DNA pellet + Centrifuged at 10,000 rpm for 5 minutes + Air dried the pellet.
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 1% of Agarose gel electrophoresis.
DNA is isolated successfully from both normal and diabetic patients blood samples.
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 18.3µl
Buffer 2.5µl
dNTPs 1µl
Forward Primers 1µl (20 pmol/µl)
Reverse Primers 1µl (20 pmol/µl)
Taq Polymerase 0.2µl
DNA sample 1µl
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
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