Biochem Oral Report PPT

7/29/2019 Biochem Oral Report PPT

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ISOLATION ANDCHARACTERIZATION

DNA

NICOLAS, Jonella JeanPOSADAS, John Arcee

SALCEDO, JenevaUY, Ryan Christopher


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Onion

We use an onion because ofits' cost, abundance and lowstarch content.

Its cell is humongous


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Nucleic Acid

A nucleic acid is a macromolecule composed ofchains of high molecular weight biopolymers ofmonomeric nucleotides. These molecules carry

genetic information or form structures within cells.

The most common nucleic acids are deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). Its functionmainly in the storage and expression of genetic

information


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Deoxyribonucleic Acid

It is the hereditary material in humans and almost allother organisms. Nearly every cells in a personsbody has the same DNA.

Most DNA is located in the cell nucleus (where it iscalled nuclear DNA)

Small amount of DNA can also be found in the

mitochondria where it is called mitochondrial DNA ormtDNA


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Composition of DNA

Sugar is Deoxyribose

Nitrogenous Bases

Phosphate Group


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Nitrogenous Bases

These nitrogenous bases hydrogen bond between opposingDNA strands to form the rungs of the "twisted ladder" or

double helix of DNA or a biological catalyst that is found inthe nucleotides.

Adenine is always paired with thymine, and guanine is alwayspaired with cytosine. Uracil is only present in RNA: replacingthymine and pairing with adenine.


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Purines

Guanine

Adenine

Pyridines

Cytosine

Thymine


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Properties of DNA

Double Stranded

The two strands run on opposite direction

The strands are complimentary and are stabilized byH-bonds between bases (G-C, A-T)

Insoluble in dilute salt solution and ethanol, solublein concentrated salt and water.

attached to a protein (histone) through ionicinteraction (nucleoprotein)


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Nomograph

It is a graphical calculating device, a two-dimensionaldiagram designed to allow the approximategraphical computation of a function.

The result is obtained by laying a straightedge acrossthe known values on the scales and reading theunknown value from where it crosses the scale forthat variable. The virtual or drawn line created by the

straightedge is called an index line or isopleth.


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Objective

To be able to isolate DNA from Onion

To be able to determined the DNA concentration and

PurityTo be able to hydrolyzed DNA by Acid Hydrolysis

To be Able to Characterized DNA that was isolated


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Methodology


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A. Isolation of DNA

from OnionChopped fresh onionsHomogenizingsolution

Ice-cold 95% ethanol

Commercial PapainCheesecloth

Blender

Erlenmeyer Flask

Kitchen Knife

Graduated Cylinder

Funnel

250ml Beaker

DNA spooler

Ice Bath


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50 ml ofhomogenizing

solution in a 250mlerlenmeyer flask

50 mlhomogenizing

solution, 60o

The first few layers ofthe onion was removedThe onion was mincedand weighed to 25g

This was added

50 mlHomogenizingsolution with onion

Solution wasstirred and let sit ina water bath for 5mins, While inwater bath ,

solution wasstirred occasionallyevery 2 mins

50 ml Homogenizingsolution with onion,still in water bath

1.500 g of Crude papain powderwas addedThe solution was kept in thewater bath for an additional 10

mins

Solution washeated to60o

50 ml Homogenizingsolution with onion and1.5 crude papain , still inwater bath

Flask wastransferred intoan ice bath for 5mins

50 ml Homogenizing solutionwith onion and 1.5 crude papain, still in ice bath

Contentswere pouredin a blender

ContentsWereblendedfor 5 mins

Homogenate

A. Isolation and Characterization of DNA


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Homogenate

Homogenate wasfiltered with 4 layers ofcheesecloth into aclean graduatedcylinder

FiltratePrecipitate


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Filtrate

Volume was

measured andtransferred to a250ml beaker

Filtrate in

250 mlbeaker

Beaker was tilted ata 45o angle

95% ethanol(2x homogenatevolume) was poured downthe the inner wall of thebeaker

Solution was leftundisturbed for 2-3minsuntil bubbling stopped

Upper layerethanolLower layer OnionLiquid

With DNA floatingon surface

DNA was collected by placingspooler just below the upperlayer of the liquid and thentwirled it in and out of the 2layers in I direction

Collected DNAWeigh and air dry

The Mixture ofethanol andonion


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B. Determination of DNA

Concentration and PurityStandard Saline Citrate

Tris-EDTA buffer

Spectophotometer


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1.0 mg DNA

Dissolve inSSC solution TE Buffer

Rest of the

DNA precipitateWas setaside forAcidHydrolysis ofDNA

B. Determination of DNA Concentration and Purity

Absorbance was read at 260 nm and 280nmAbsorbance ratio was calculate usingthe equation A260 / A280

A260 = Absorbanceratio of 260nmA280= Absorbanceratio of 280nm


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C. Acid Hydrolysis of

DNA1M sodium hydroxide1M HCl

Medium sized test tube

Boiling water bath

Distilled water

Marble

Filter paper

Graduated cylinder

Funnel

C A id H d l i f DNA


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DNAPrecipitate

DNA was mixed with1.0ml of HCl in a mediumsized test tube

DNA precipitate

mixed with 1.0ml of60% HClO in mediumsi sized test tube

The test tube was coveredwith a marble and

heated at 100o for 60 minsContents of the test tubewas agitated occasionally

Medium sized test tubecontaining solution

Test tube was removedfrom the water bathAllowed to cool in room

temperature

2.5 ml of distilledwater was addedNeutralized with 1M

Sodium Hydroxide

Medium sized Test tube

containing solution + 2.5ml ofdistilled water, Neutral

Particulate matter?NO YES

Store DNA hydrolyzatein refrigerator for DNAcharacterization

Hydrolyzate wasfiltered

Distilled water wasadded to 3ml

3ml of Filteredsoln

C. Acid Hydrolysis of DNA


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D. Chemical

Characterization of DNAD.1 Test for Deoxyribose or Dische ReactionDNA Hydrolyzate

Diphenylamine

Standard Deoxyribose Solution

D.2 Test for Phosphate

DNA Hydrolyzate

Conc. HNO3Conc. H2SO4


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D. Chemical

Characterization of DNAD.3 Test for Purines / Murexide testDNA Hydrolyzate

Conc. HNO3

10% KOHStandard Guanine or Adenine Solution

D.4 Test for Pyrimidines/Wheeler Johnsons Test

DNA Hydrolyzate

Bromine WaterBarium Hydroxide

Standard cytosine or uracil solution


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D. Chemical Characterization of DNA

D1. Test for Deoxyribose and Dische Reaction

0.5 ml of DNAHydrolyzate

0.5ml ofDeoxyribosestandard solution

1.5ml of diphenylamine was added

0.5ml of Deoxyribosestandard solution + 1.5ml of Diphenylamine

0.5 ml of DNAHydrolyzate +1.5 ml ofdiphenylamine

Heated in a boiling waterbath for 10 mins

0.5 ml of DNAHydrolyzate +1.5 ml ofdiphenylamine

0.5 ml of DNAHydrolyzate + 1.5 ml ofdiphenylamine

Results were

observed


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D2. Test for Phosphate

1 ml of DNAHydrolyzate

1ml of Phosphatestandard solution

1ml of conc H2SO4 was added

0.5ml of Phosphatestandard solution +1mlof conc H2SO4

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4

0.5 ml of conc HNO3 wasadded

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3

0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 +0.5ml ofconc HNO3

l f DNA


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1ml of Phosphatestandard solution1ml Distilled water was added

Heated for 5 mins in a boiling water bath

1ml of AmmoniumMolybdate solution was

added

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3

0.5 ml of Phosphatestandard solution +1mlof concH2SO4 +0.5ml of conc

HNO3

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilledwater

0.5 ml of Phosphatestandard solution +1ml

of conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilledwater

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilledwater + 1ml AmmoniumMolybdate solution

0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilledwater + 1ml Ammonium

Molybdate solution


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1ml of Phosphatestandard solution

0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilled

water + 1ml AmmoniumMolybdate solution

0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilled

water + 1ml AmmoniumMolybdate solution

Diluted to 10ml with waterSolutiton was let stand for 10 mins

Color of solution andprecipitate formedwas observed


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D3. Test for Purines /Murexide Test

DNA Hyrdolyzatein smallevaporating dish

Standard adenine or guaninesolution in small evaporatingdish

A few drops of conc. Nitric acid, was addedEvaporated to dryness in the fume hood

DNA Hyrdolyzate +Conc. Nitric acid insmall evaporatingdish

Standard adenine or guaninesolution + Conc. Nitric acid insmall evaporating dish

Upon moistening with 10%KOH , theyellow residue became red and thenwas seen with a purplish red hueA few drops of water was addedAnd observations were recorded


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Light yellow Solution Light yellow Solution

Barium Hydroxide was added inexcess.Tested using litmus paper, to verifyIf Basic

Results were then observed


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Results and Discussion


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Isolation of DNA fromOnion


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GRP.Weight ofOnion (g)

Volume offiltrate

(mL)Description of DNA

Weight of air-dried precipitate

(g)

1 25.0377g 40mL Clear yellow solution 0.1241g

2 24.1387g 39mL Clear yellow solution 0.0542g

3 25.07g 45mLTurbid yellowsolution

0.35g

4 25.13g 41mL Clear yellowishsolution

0.1839g

5 26.42g 45mLTurbid light yellowsolution

0.3727


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GRP.Weight ofOnion (g)

Volume offiltrate

(mL)Description of DNA

Weight of air-dried precipitate

(g)

6 25.63g 41mLTurbid of yellowishsolution

0.1938g

7 25.01g 38mLTurbid yellowishsolution 0.24g

8 25.41g 41mL Light yellow solution 0.1102g

9 25.05g 39mL Turbid yellowishsolution

0.0704g

10 23.98g 36mL Light yellow solution 0.2733g


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HeatingSoftens lipid membranes of the cell

Enhances the function of homogenizing solution

Breakdown of cell membrane

Temperature kept at 60C

For maximum activity of the enzyme papain in DNA

deproteinationDNA is degraded at 75C


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Homogenizingsolution

Destroys the cell and nuclear membranes of the onioncells

4 important components:

Sodium Dodecyl Sulfate (SDS)

Sodium citrate

Ethylenediamine tetracetic acid(EDTA)

Sodium chloride


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Sodium Dodecyl Sulfate (SDS)

anionic biological detergent

break down and effectively emulsify the lipid and proteincomponents of the cell

disruption of polar interactions that keep the membrane together

Sodium citrate

chelating agent

causes cellular debris (degraded cell membranes, etc.) toprecipitate for easy filtration

Sodium chloridehelps the DNA to precipitate out of the solution

shields the negative charges of the phosphate group in the DNAbackbone


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Crude Papain Powder

Ethylenediamine tetracetic acid (EDTA)

chelating agentbinds to Mg which is needed for Dnase activity

*Dnase is an enzyme that degrades DNA (undesirable)

For deproteination

Papain enzyme that breaks peptide bonds attach to DNA Optimal temperature range: 60C


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CoolingSlows down DNA breakdown

Swirling even cooling of solution

Blending Further breakdown of cell membranes

45 sec only in order not to destroy DNA molecules


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FiltrationCheesecloth - traps the precipitated cell debris while thesoluble DNA passes through

Ice-cold 95% ethanol Precipitates the DNA

Causes the other components of the filtrate to stay in thesolution


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Determination of DNA

Concentration and Purity


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Group # 260nm 280nm Protein

concentration

Nucleic Acid

Concentration

Absorbance

Ratio

1 0.345 0.372 0.3 mg/mL 8g/mL 0.927

2 0.371 0.373 0.29 mg/mL 7g/mL 0.995

3 0.638 0.523 0.25mg/mL 20g/mL 1.22

4 0.220 0.227 0.22 mg/mL 5g/mL 0.97

5 0.591 0.562 0.4mg/mL 15g/mL 1.05


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Group # 260nm 280nm Protein

concentration

Nucleic Acid

Concentration

Absorbance

Ratio

6 0.183 0.201 0.22 mg/mL g/mL 0.910

7 0.242 0.259 0.30 mg/mL 5.5g/mL 0.9343

8 0.527 0.523 0.35 mg/mL 14.5g/mL 1.0076

9 0.631 0.630 0.48 mg/mL 16g/mL 1.0016

10 1.525 1.335 0.68 mg/mL 50g/mL 1.142


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Light absorbance of DNA

260 nm

Purines and pyrimidines are detected

Light absorbance of proteins

280 nm

Aromatic amino acids are detectedTyrosine, tryptophan and phenylalanine

Good DNA sample:

1.7-2.0 absorbance ratio (A260/A280)Outside this range, DNA solution is contaminated

Lower than expected range: protein contaminated

Higher than expected range: RNA contaminated


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Acid Hydrolysis ofDNA


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Addition of 1M HCl and heating at 100C

To breakdown the double helix structure of DNA

Strong acids at high temperature can break:Phosphodiester bonds

o separates phosphate group from deoxyribose sugar andnitrogenous base complex

-N-glycosidic bonds

o Separates nitrogenous bases from deoxyribose sugar

Hydrogen bonds

o Separates nitrogenous bases pairs

Neutralized with 1M NaOHo acidic condition can affect the results of the characterization

tests


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Chemical

Characterization of DNA


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Standard


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Group # Test forDeoxyribose

Test forPhosphate

Test for Purines Test forPyrimidines

1 Dark purple

solution

Clear Solution

YellowPrecipitate

Yellow Liquid

Orange SolutionRed OrangeResidue

Clear solution

Purple precipitate

2 Dark blue solution Turbid yellowsolutionYellow

precipitate

Yellow precipitateYellow solutionRed Orange

residue

Purple solutionPurple precipitate

3 Dark purplesolution

Clear colorlesssolutionYellowprecipitate

Yellow precipitateYellow solutionRed orangeresidue

Clear colorlesssolution

4 Dark purple

solution

Clear yellow

solutionYellowprecipitate

Yellow precipitate

Yellow solutionRed orangeprecipitate

Purple Solution

Purple precipitate

5 Dark purplesolution

Yellowprecipitate

Yellow precipitateYellow solutionRed precipitate

Purple residue


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Test forPhosphate


6 Dark blue solution Clear yellow

solutionWhiteprecipitate

Red orange solid

formedYellow precipitate

Purple color

solution

7 Dark bluesolution

Light yellowsolutionYellowprecipitate

Clear solutionYellow residueRed residue

Violet residue

PurpleprecipitateColorlesssolution

8 Dark violet solution Yellowprecipitate

White residueYellow solutionBrown yellowsolutionYellow residue

Purple precipitateClear colorlesssolution

9 Dark PurpleSolution

YellowprecipitateColorlesssolution

White solutionYellow residueRed residue

Purple PrecipitateColorless Solution

10 Dark Purple opaquesolution

Clear solutionYellow

precipitate

Clear solutionPurple precipitate

Red OrangePrecipitate


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DNA Hydrolyzate


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Test forPhosphate


1 Light silver solution

White precipitate

Clear solution

WhitePrecipitate

Light yellow

solutionLight brownresidue

Clear solution

White precipitate

2 Clear blue solution Clear light yellowsolutionLight yellowprecipitate

Clear solutionBrown solutionLight yellowishbrown precipitate

Light yellowsolutionWhite precipitate

3 Clear light greensolution

Clear colorlesssolution

Colorless solutionBrown solutionLight yellowsolutionWhite precipitate

Light yellowsolutionWhite precipitate

4 Greenish graysolution

Clear solution

Whiteprecipitate

Brown precipitate

Brown solutionBrown precipitate

Yellowish solution

with precipitate

5 Turbid light bluesolution

Turbid lightyellow solution

Colorless residueBrown solutionBrown residue

Turbid whitesolution


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Test forPhosphate


6 Light blue solution Clear light yellow

solution

White sticky solid

formWhite powder

Colorless solution

7 Light Blue solutionWhite particles

Clear colorlesssolutionNo precipitate

Clear solutionYellow residueYellowish solution

Colorless solutionWhite particles

8 Turbid Solution Clear colorlesssolution

Yellow residueRed solutionPurplish redresidueYellow solution

Clear colorlesssolutionWhite precipitate

9 Clear colorlesssolution

Clear colorless

solution

Colorless solution

Yellow residue

Clear colorless

Solution

10 Light Blue TurbidSolution

No precipitateClear solution

White precipitateClear solution

Light brownprecipitate

T t f D ib


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Test for Deoxyribose orDische Test

POSITIVE RESULT

Blue colored solution

PRINCIPLEDehydration of deoxypentose to

hydroxylaevulinic aldehyde

Complexation of hydroxylaevulinic aldehyde withdiphenylamine (blue solution)

Intensity of blue color is proportional to DNA concentration


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2-deoxyribose

Aldopentose of DNA (RNA- ribose)

Lack oxygen atom at C2


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Test for PhosphatePOSITIVE RESULT

Yellow precipitate

PRINCIPLE

Complexation of phosphate with ammonium molybdate inan acidic medium to form phosphoammonium molybdate(yellow precipitate )

H3PO4 + HNO3 + 12 (NH4)2MoO4(NH4)3PO4.12MoO3

T t f P i


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Test for Purines orMurexide Test

POSITIVE RESULT

Red precipitate

PRINCIPLE

Oxidation of purine by conc. HNO3 forming dialuric acidand alloxan (yellow precipitate)

Condensation leading to formation of alloxanthineAlloxanthine reacts with 10% KOH to form murexide (redprecipitate)


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Test for Pyrimidines

POSITIVE RESULT

Purple color

PRINCIPLE

Bromination of pyrimidines at C5 to producedibromoxhydrouracil (yellow solution)

Alkaline hydrolysis caused by addition of Ba(OH)2 toproduce isodialuric acid

Rearrangement to form dialuric acid and formationof its barium salt (purple precipitate)


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Negative for thymine because it is methylated at C5.


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Conclusion

Many measures have to be taken into account in order to completelyisolate DNA from onion. There are three basic procedure used toisolate DNA: homogenization, deproteination and precipitation.

Purity of isolated DNA can be determined by its protein and nucleic

acid light absorbance with the use of nomograph.Acid hydrolysis was used to hydrolyzed DNA (break the bonds of theDNA) for it to be characterized.

The test for deoxyribose produce a brownish solution, the test forphosphate produce turbid solution with yellow precipitate, the test

for purine produce a red residue, and the test for pyrimidinesproduce purple precipitate which is positive to all the test. However,some of the results from the experiment had a negative resultsbecause of errors. The source of error comes from incorrect use ofreagents.

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Biochem Oral Report PPT