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NICOLAS, Jonella Jean POSADAS, John Arcee SALCEDO, Jeneva UY, Ryan Christopher
We use an onion because of its' cost, abundance and low starch content. Its cell is humongous
A nucleic acid is a macromolecule composed of chains of high molecular weight biopolymers of monomeric nucleotides. These molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Its function mainly in the storage and expression of genetic information
It is the hereditary material in humans and almost all other organisms. Nearly every cells in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA) Small amount of DNA can also be found in the mitochondria where it is called mitochondrial DNA or mtDNA
Composition of DNA
Sugar is Deoxyribose Nitrogenous Bases
Adenine is always paired with thymine. and guanine is always paired with cytosine. .Nitrogenous Bases These nitrogenous bases hydrogen bond between opposing DNA strands to form the rungs of the "twisted ladder" or double helix of DNA or a biological catalyst that is found in the nucleotides. Uracil is only present in RNA: replacing thymine and pairing with adenine.
Purines Guanine Adenine Pyridines Cytosine Thymine .
attached to a protein (histone) through ionic interaction (nucleoprotein) . A-T) Insoluble in dilute salt solution and ethanol.Properties of DNA Double Stranded The two strands run on opposite direction The strands are complimentary and are stabilized by H-bonds between bases (G-C. soluble in concentrated salt and water.
Nomograph It is a graphical calculating device. The result is obtained by laying a straightedge across the known values on the scales and reading the unknown value from where it crosses the scale for that variable. The virtual or drawn line created by the straightedge is called an index line or isopleth. a two-dimensional diagram designed to allow the approximate graphical computation of a function. .
Objective To be able to isolate DNA from Onion To be able to determined the DNA concentration and Purity To be able to hydrolyzed DNA by Acid Hydrolysis To be Able to Characterized DNA that was isolated .
A. Isolation of DNA from Onion Chopped fresh onions Homogenizing solution Ice-cold 95% ethanol Commercial Papain Cheesecloth Blender Erlenmeyer Flask Kitchen Knife Graduated Cylinder Funnel 250ml Beaker DNA spooler Ice Bath .
still in water bath Flask was transferred into an ice bath for 5 mins 50 ml Homogenizing solution with onion and 1. still in water bath 50 ml Homogenizing solution with onion 50 ml Homogenizing solution with onion and 1. Isolation and Characterization of DNA 50 ml of homogenizing solution in a 250ml erlenmeyer flask Solution was heated to 60o 50 ml homogenizing solution. While in water bath .500 g of Crude papain powder was added The solution was kept in the water bath for an additional 10 mins 50 ml Homogenizing solution with onion. still in ice bath Contents were poured in a blender Contents Were blended for 5 mins Homogenate .5 crude papain . solution was stirred occasionally every 2 mins The first few layers of the onion was removed The onion was minced and weighed to 25g This was added 1. 60o Solution was stirred and let sit in a water bath for 5 mins.A.5 crude papain .
Homogenate Homogenate was filtered with 4 layers of cheesecloth into a clean graduated cylinder Precipitate Filtrate .
Filtrate Volume was measured and transferred to a 250ml beaker The Mixture of ethanol and onion Collected DNA Weigh and air dry DNA was collected by placing spooler just below the upper layer of the liquid and then Filtrate in twirled it in and out of the 2 250 ml layers in I direction beaker 95% ethanol(2x homogenate Upper layer – volume) was poured down ethanol the the inner wall of the Lower layer – Onion beaker Liquid With DNA floating on surface Beaker was tilted at a 45o angle Solution was left undisturbed for 2-3mins until bubbling stopped .
B. Determination of DNA Concentration and Purity Standard Saline Citrate Tris-EDTA buffer Spectophotometer .
B. Determination of DNA Concentration and Purity Rest of the DNA precipitate Was set aside for Acid Hydrolysis of DNA 1.0 mg DNA SSC solution TE Buffer Dissolve in A260 = Absorbance ratio of 260nm A280= Absorbance ratio of 280nm Absorbance was read at 260 nm and 280 nm Absorbance ratio was calculate using the equation A260 / A280 .
Acid Hydrolysis of DNA 1M sodium hydroxide 1M HCl Graduated cylinder Funnel Medium sized test tube Boiling water bath Distilled water Marble Filter paper .C.
C. Neutral Medium sized test tube containing solution Test tube was removed from the water bath Allowed to cool in room temperature 2.0ml of 60% HClO in medium si sized test tube The test tube was covered with a marble and heated at 100o for 60 mins Contents of the test tube was agitated occasionally Store DNA hydrolyzate in refrigerator for DNA characterization 3ml of Filtered soln Hydrolyzate was filtered Distilled water was added to 3ml Particulate matter? NO YES Medium sized Test tube containing solution + 2.0ml of HCl in a medium sized test tube DNA precipitate mixed with 1. Acid Hydrolysis of DNA DNA Precipitate DNA was mixed with 1.5 ml of distilled water was added Neutralized with 1M Sodium Hydroxide .5ml of distilled water.
H2SO4 . HNO3 Conc.1 Test for Deoxyribose or Dische Reaction DNA Hydrolyzate Diphenylamine Standard Deoxyribose Solution D.2 Test for Phosphate DNA Hydrolyzate Conc. Chemical Characterization of DNA D.D.
4 Test for Pyrimidines/Wheeler Johnson’s Test DNA Hydrolyzate Bromine Water Barium Hydroxide Standard cytosine or uracil solution .D. HNO3 10% KOH Standard Guanine or Adenine Solution D.3 Test for Purines / Murexide test DNA Hydrolyzate Conc. Chemical Characterization of DNA D.
5ml of Deoxyribose standard solution + 1.5 ml of DNA Hydrolyzate + 1. Test for Deoxyribose and Dische Reaction 0.5 ml of DNA Hydrolyzate +1.5 ml of diphenylamine .5 ml of diphenylamine Results were observed 0.5ml of Deoxyribose standard solution 1. Chemical Characterization of DNA D1.5 ml of DNA Hydrolyzate 0.5ml of diphenylamine was added 0.5 ml of diphenylamine 0.5 ml of Diphenylamine Heated in a boiling water bath for 10 mins 0.D.5 ml of DNA Hydrolyzate +1.
5 ml of conc HNO3 was added 0.5ml of conc HNO3 0. Test for Phosphate 1 ml of DNA Hydrolyzate 1ml of conc H2SO4 was added 0.5 ml of DNA Hydrolyzate +1ml of conc H2SO4 + 0.5 ml of DNA Hydrolyzate +1ml of conc H2SO4 1ml of Phosphate standard solution 0.D2.5 ml of Phosphate standard solution +1ml of conc H2SO4 +0.5ml of conc HNO3 .5ml of Phosphate standard solution +1ml of conc H2SO4 0.
5 ml of DNA Hydrolyzate +1ml of conc H2SO4 + 0.0.5ml of conc HNO3 1ml of Phosphate 1ml Distilled water was added standard solution Heated for 5 mins in a boiling water bath 0.5ml of conc HNO3 +1ml distilled water + 1ml Ammonium Molybdate solution 1ml of Ammonium Molybdate solution was added 0.5 ml of DNA Hydrolyzate +1ml of conc H2SO4 + 0.5 ml of DNA Hydrolyzate +1ml of conc H2SO4 + 0.5ml of conc HNO3 0.5 ml of Phosphate standard solution +1ml of conc H2SO4 +0.5ml of conc HNO3 +1ml distilled water 0.5ml of conc HNO3 +1ml distilled water + 1ml Ammonium Molybdate solution .5 ml of Phosphate standard solution +1ml of conc H2SO4 + 0.5 ml of Phosphate standard solution +1ml of conc H2SO4 + 0.5ml of conc HNO3 +1ml distilled water 0.
5ml of conc HNO3 +1ml distilled water + 1ml Ammonium Molybdate solution 0.5ml of conc HNO3 +1ml distilled water + 1ml Ammonium 1ml of Phosphate Molybdate solution standard solution Diluted to 10ml with water Solutiton was let stand for 10 mins Color of solution and precipitate formed was observed .5 ml of Phosphate standard solution +1ml of conc H2SO4 + 0.0.5 ml of DNA Hydrolyzate +1ml of conc H2SO4 + 0.
D3. Test for Purines /Murexide Test DNA Hyrdolyzate in small evaporating dish Standard adenine or guanine solution in small evaporating dish A few drops of conc. Nitric acid. Nitric acid in small evaporating dish Upon moistening with 10%KOH . the yellow residue became red and then was seen with a purplish red hue A few drops of water was added And observations were recorded . Nitric acid in small evaporating dish Standard adenine or guanine solution + Conc. was added Evaporated to dryness in the fume hood DNA Hyrdolyzate + Conc.
D4.5 ml of Start cytosine or uracil solution Treated with with an excess bromine water until solution turned yellow Yellow Solution Yellow Solution Solution was boiled until it became light yellow to colorless. Test for Pyrimidines/ Wheeler Johnson’s Test 0.5 ml of DNA Hydrolyzate 0. done in the hood This was done to remove excess bromine Light yellow Solution Light yellow Solution .
Tested using litmus paper. to verify If Basic Light yellow Solution Results were then observed .Light yellow Solution Barium Hydroxide was added in excess.
Results and Discussion .
Isolation of DNA from Onion .
42g 45mL 0.07g 45mL Turbid yellow solution Clear yellowish solution Turbid light yellow solution 0.35g 4 25.GRP.1241g 1 25.1387g 39mL Clear yellow solution 0.3727 .0377g 2 24. Weight of Onion (g) Volume of filtrate (mL) 40mL Weight of airDescription of DNA dried precipitate (g) Clear yellow solution 0.0542g 3 25.13g 41mL 0.1839g 5 26.
1938g 6 25.98g 36mL 0.05g 39mL 0. Weight of Onion (g) Volume of filtrate (mL) 41mL Weight of airDescription of DNA dried precipitate (g) Turbid of yellowish solution Turbid yellowish solution 0.41g 41mL Light yellow solution Turbid yellowish solution Light yellow solution 0.0704g 10 23.GRP.01g 38mL 0.1102g 9 25.2733g .24g 8 25.63g 7 25.
Heating Softens lipid membranes of the cell Enhances the function of homogenizing solution Breakdown of cell membrane Temperature kept at 60°C For maximum activity of the enzyme papain in DNA deproteination DNA is degraded at 75°C .
Destroys the cell and nuclear membranes of the onion cells 4 important components: Sodium Dodecyl Sulfate (SDS) Homogenizing solution Sodium citrate Ethylenediamine tetracetic acid(EDTA) Sodium chloride .
etc.Sodium Dodecyl Sulfate (SDS) anionic biological detergent break down and effectively emulsify the lipid and protein components of the cell disruption of polar interactions that keep the membrane together Sodium citrate chelating agent causes cellular debris (degraded cell membranes.) to precipitate for easy filtration Sodium chloride helps the DNA to precipitate out of the solution shields the negative charges of the phosphate group in the DNA backbone .
Ethylenediamine tetracetic acid (EDTA) chelating agent binds to Mg which is needed for Dnase activity *Dnase is an enzyme that degrades DNA (undesirable) Crude Papain Powder For deproteination Papain – enzyme that breaks peptide bonds attach to DNA Optimal temperature range: 60°C .
Cooling Slows down DNA breakdown Swirling – even cooling of solution Blending Further breakdown of cell membranes 45 sec only – in order not to destroy DNA molecules .
traps the precipitated cell debris while the soluble DNA passes through Ice-cold 95% ethanol Precipitates the DNA Causes the other components of the filtrate to stay in the solution .Filtration Cheesecloth .
Determination of DNA Concentration and Purity .
371 0.373 0.22 mg/mL 0.995 1.Group # 260nm 280nm Protein concentration Nucleic Acid Concentration Absorbance Ratio 1 2 3 4 5 0.523 0.97 1.29 mg/mL 0.05 .3 mg/mL 0.591 0.562 0.4mg/mL 8 μg/mL 7μg/mL 20μg/mL 5 μg/mL 15μg/mL 0.372 0.220 0.638 0.25mg/mL 0.22 0.227 0.345 0.927 0.
5 μg/mL 14.242 0.0016 10 1.5 μg/mL 16 μg/mL 0.183 0.525 1.631 0.Group # 260nm 280nm Protein concentration Nucleic Acid Concentration Absorbance Ratio 6 7 8 9 0.68 mg/mL 50μg/mL 1.910 0.335 0.142 .9343 1.527 0.201 0.35 mg/mL 0.30 mg/mL 0.0076 1.630 0.523 0.48 mg/mL μg/mL 5.259 0.22 mg/mL 0.
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 .Light absorbance of DNA 260 nm Purines and pyrimidines are detected Light absorbance of proteins 280 nm Aromatic amino acids are detected Tyrosine. tryptophan and phenylalanine Good DNA sample: 1.
Acid Hydrolysis of DNA .
Addition of 1M HCl and heating at 100°C To breakdown the double helix structure of DNA Strong acids at high temperature can break: Phosphodiester bonds o separates phosphate group from deoxyribose sugar and nitrogenous base complex β-N-glycosidic bonds o Separates nitrogenous bases from deoxyribose sugar Hydrogen bonds o Separates nitrogenous bases pairs – Neutralized with 1M NaOH o acidic condition can affect the results of the characterization tests .
Chemical Characterization of DNA .
Group # Test for Deoxyribose Test for Phosphate Test for Purines Test for Pyrimidines 1 Dark purple solution Clear Solution Yellow Precipitate Turbid yellow solution Yellow precipitate Clear colorless solution Yellow precipitate Clear yellow solution Yellow precipitate Yellow precipitate Yellow Liquid Orange Solution Red Orange Residue Clear solution Purple precipitate 2 Dark blue solution Yellow precipitate Yellow solution Red Orange residue Yellow precipitate Yellow solution Red orange residue Yellow precipitate Yellow solution Red orange precipitate Purple solution Purple precipitate 3 Dark purple solution Clear colorless solution 4 Dark purple solution Purple Solution Purple precipitate 5 Dark purple solution Yellow precipitate Yellow solution Red precipitate Purple residue .
Group # Test for Deoxyribose Test for Phosphate Test for Purines Test for Pyrimidines 6 Dark blue solution Clear yellow solution White precipitate Red orange solid formed Yellow precipitate Clear solution Yellow residue Red residue Violet residue White residue Yellow solution Brown yellow solution Yellow residue White solution Yellow residue Red residue Clear solution Purple precipitate Purple color solution 7 Dark blue solution Light yellow solution Yellow precipitate Yellow precipitate Purple precipitate Colorless solution Purple precipitate Clear colorless solution 8 Dark violet solution 9 Dark Purple Solution Yellow precipitate Colorless solution Clear solution Yellow precipitate Purple Precipitate Colorless Solution 10 Dark Purple opaque solution Red Orange Precipitate .
DNA Hydrolyzate .
Group # Test for Deoxyribose Light silver solution White precipitate Test for Phosphate Clear solution White Precipitate Clear light yellow solution Light yellow precipitate Clear colorless solution Test for Purines Test for Pyrimidines Clear solution White precipitate 1 Light yellow solution Light brown residue Clear solution Brown solution Light yellowish brown precipitate Colorless solution Brown solution Light yellow solution White precipitate Brown precipitate Brown solution Brown precipitate Colorless residue Brown solution Brown residue 2 Clear blue solution Light yellow solution White precipitate Light yellow solution White precipitate 3 Clear light green solution 4 Greenish gray solution Clear solution White precipitate Turbid light yellow solution Yellowish solution with precipitate 5 Turbid light blue solution Turbid white solution .
Group # Test for Deoxyribose Light blue solution Test for Phosphate Test for Purines Test for Pyrimidines Colorless solution 6 Clear light yellow White sticky solid solution form White powder 7 Light Blue solution White particles Clear colorless solution No precipitate Clear colorless solution Clear solution Yellow residue Yellowish solution Yellow residue Red solution Purplish red residue Yellow solution Colorless solution White particles 8 Turbid Solution Clear colorless solution White precipitate 9 Clear colorless solution Clear colorless solution Colorless solution Yellow residue Clear colorless Solution 10 Light Blue Turbid Solution No precipitate Clear solution White precipitate Clear solution Light brown precipitate .
Test for Deoxyribose or Dische Test POSITIVE RESULT Blue colored solution PRINCIPLE Dehydration of deoxypentose to hydroxylaevulinic aldehyde Complexation of hydroxylaevulinic aldehyde with diphenylamine (blue solution) Intensity of blue color is proportional to DNA concentration .
2-deoxyribose Aldopentose of DNA (RNA.ribose) Lack oxygen atom at C2 .
Test for Phosphate POSITIVE RESULT Yellow precipitate PRINCIPLE Complexation of phosphate with ammonium molybdate in an acidic medium to form phosphoammonium molybdate (yellow precipitate ) H3PO4 + HNO3 + 12 (NH4)2MoO4 (NH4)3PO4.12MoO3 .
HNO3 forming dialuric acid and alloxan (yellow precipitate) Condensation leading to formation of alloxanthine Alloxanthine reacts with 10% KOH to form murexide (red precipitate) .Test for Purines or Murexide Test POSITIVE RESULT Red precipitate PRINCIPLE Oxidation of purine by conc.
Test for Pyrimidines POSITIVE RESULT Purple color PRINCIPLE Bromination of pyrimidines at C5 to produce dibromoxhydrouracil (yellow solution) Alkaline hydrolysis caused by addition of Ba(OH)2 to produce isodialuric acid Rearrangement to form dialuric acid and formation of its barium salt (purple precipitate) .
.Negative for thymine because it is methylated at C5.
Acid hydrolysis was used to hydrolyzed DNA (break the bonds of the DNA) for it to be characterized. The test for deoxyribose produce a brownish solution. deproteination and precipitation. and the test for pyrimidines produce purple precipitate which is positive to all the test. Purity of isolated DNA can be determined by its protein and nucleic acid light absorbance with the use of nomograph. The source of error comes from incorrect use of reagents.Conclusion Many measures have to be taken into account in order to completely isolate DNA from onion. However. . There are three basic procedure used to isolate DNA: homogenization. some of the results from the experiment had a negative results because of errors. the test for purine produce a red residue. the test for phosphate produce turbid solution with yellow precipitate.
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