Biochemistry Laboratory Formal Report CHEMISTRY
Isolation and characterization of 600L deoxyribonucleic acid (DNA) from Allium from Allium cepa EXPT 08
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Marj Marj Hipoli Hipolito, to, Von Von Gabrie Gabriell S. Tan, Tan, Reina Reina Justi Justina na P. Tolet Tolete, e, Clari Clarisse sse Anne D. Yung, Angelo D. Villaroman* Department of Chemistry, College of Science Date submitted: 23 November 2015
*Corresponding author; e-mail:
[email protected] [email protected]
Abstract The purpose of thi this experiment is to isolate and character terize deox deoxyr yrib ibon onuc ucle leic ic acid acid (DNA (DNA)) from from onio onion n ( Allium cepa) cell cells s. In the the expe experi rime ment nt,, isol isolat atio ion n of onio onion n cell cells’ s’ DNA DNA was was unde underg rgon one e thro throug ugh h homoge homogeniz nizati ation on proce process ss using using sodium sodium dodec dodecyl yl sulfa sulfate te (SDS), (SDS), sodium sodium citrate (NaC6H5O7) and sodium chloride (NaCl). Afterwhich, the isolate is tested tested for its concentr concentration ation and purity purity (via spectrop spectrophotom hotometri etric c analysi analysis) s) and characte characterize rized d using using chemica chemicall tests tests (Murexide (Murexide test, test, Dische Dische reaction reaction,, Wheeler-Johnson test, and phosphate test). The results obtained were: no DNA were extracted based form absorbance reading and a positive result for Wheeler-Johnson Test only.
Keywords: nucleic acids, deoxyribonucleic acid (DNA), spectrophotometry Introduction
Life will never be possible without reproduction. The propagation and multiplication of life is ultimately dependent on the so-called genes. genes. The main component of these genes are nucleic acids which are of two types: deoxyribonucleic acids or, DNA, (the focus of this this expe experi rime ment nt)) and and ribonu ribonuclei cleic c acids acids or or, RNA. Nuc Nucle leic ic acid acids s are are esse essent ntia iall biomolecules that are responsible for the transfer and storage of genetic information
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Biochemistry Laboratory Formal Report from generation to generation. It is composed of monomers called nucleotides which consist of three (3) parts: a ribose sugar , a phosphate group, group , and a nitrogenous base. base . Figure 1 shows the general structure of a nucleotide. The sugar component is an aldopentose, a β-D-ribose (in RNA) Figure 1. General structure of a nucleotide
or a β-D-deoxyribose (in DNA) (Boyer, 2012). In DNA, the hydr hydrox oxyl yl grou group p (-OH (-OH)) in C-2 C-2 posi positi tion on of the the suga sugarr is
replaced by a hydrogen, thus, deoxygenated . The phosphate group, on the other hand, makes the nucleotide negatively charged and enables it to bond to other nucleotides (polymer (polymerizat ization) ion).. Nucleot Nucleotides ides are held together together by a 3’,5’-phosphodiester 3’,5’-phosphodiester bonds and experience directionality -- one end of the chain has a 3’-hydroxyl (or phosphate) group and the other other end has a 5’-hy 5’-hydro droxy xyll (or phosph phosphate ate)) group group.. Both Both the sugar sugar and the phosphate groups constitute the common, invariant region of the nucleotide (referred to as the backbone)(Boyer, 2012). On the other hand, the nitrogenous base is the variable region of the compo compoun und. d. There There are are 2 types types of nitro nitrogen genous ous bases bases:: pyrimidines and purines. purines. Both Both are are name named d such such for for they they rese resemb mble le eith either er a pyramidine or a purine structure. Pyramidine bases are singlering aromatic compound which includes cytosine, thymine, and uracil. Cytosine is present in both DNA or RNA. Thymine, on the other hand, is substituted for uracil in DNA. Uracil occurs only in RNA. Purine bases, however, are double-ring aromatic
Figure 2. Purines and pyrimidines pyrimidines
compounds. It includes adenine and adenine and guanine, both guanine, both of which is found in DNA and in RNA (Campbell & Farell, 2012). Figure 2 shows the five (5) heterocyclic bases.
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Biochemistry Laboratory Formal Report Native Native DNA exists as two, two, compleme complementar ntary, y, antipar antiparalle allell strands strands arranged arranged in a double double helix held by noncovalent bonding. The two strands of DNA wound around each other with with the bases bases insid inside e and and the sugarsugar-pho phosp sphat hate e backb backbone one on the the outsid outside. e. The The most most important and significant feature of the double helix is its complementary base pairing. In each pair, a purine and a pyrimidine pairs together, thus, adenine (A) always pairs with thymine (T), and guanine (G) always pairs with cytosine (C). The forces that holds the the doub double le heli helix x are: are: the the hydrogen hydrogen bonds bonds of of the the base base pair pairs, s, and and the the hydrophobic interactions and van and van der Waals forces between ‘stacked bases’ (Boyer, 2012). Today, there are a lot of isolation and characterization methods of nucleic acids. The isolatio isolation n techniq techniques ues used in this experime experiment nt are through through mechanical, mechanical, enzymatic, enzymatic, and chemical means chemical means.. Mechanical method employs physical treatment like grinding, stirring, chopping chopping,, and other disruptiv disruptive e procedu procedures. res. On one hand, hand, enzymati enzymatic c involve involves s using using enzyme enzymes s to hydro hydroly lyze ze certai certain n bonds bonds e.g. e.g. prote protease ases. s. Lastly Lastly,, using using chemic chemicals als like like detergents disrupt the lipid components of the cell. Moreover, characterization of nucleic acid acid in this this exper experime iment nt involv involves es spec spectro troph photo otomet metric ric analy analysis sis,, which which runs runs under under the principle of light absorbance (chromophores; in DNA conjugated bases (A,T,C,G)) and chemical chemical tests (qualit (qualitativ ative) e) like Dische, Dische, Murexide, Murexide, Wheeler-Johnson, Wheeler-Johnson, and phosphate test . Dische test detects the presence of sugars in a solution. A blue solution indicates a positi positive ve resul resultt to this this test. test. Murex Murexide ide test test ident identifi ifies es presen presence ce of caffe caffeine ine and purine purine derivatives. A pink residue is positive for this test. Wheeler-Johnson test detects uracil or cytosine in a solution. A purple or violet-blue color is positive in this test (Wheeler, H.L., & Johnson, T.B, 1907). Lastly phosphate test detects presence of phosphate in a solution, if phosphate ions are present, a bright yellow precipitate is observed. The objective of this experiment is to isolate and characterize DNA from onion cells. 3
Biochemistry Laboratory Formal Report Results and discussion discussion
The experiment is divisible into three parts as already said previously -- isolation, test for concentration and purity, and chemical characterization. I. Isolation of DNA
Three (3) methods are involved in extracting the DNA namely, mechanical, enzymatic, and chemical . The The init initia iall trea treatm tmen entt to the the onio onion, n, cutt cuttin ing, g, is part part of the the mech mechan anic ical al degradat degradation ion which which helps helps in the preliminary preliminary disruption disruption of the onion cell. cell. Howeve However, r, too much mechanical stress can induce the activity of DNase, which if present, would cut the DNA into smaller fragments; this would not allow the DNA to be spooled. Also, the heat treatment softens the phospholipid in the cell membrane (easier to degrade) and denatures DNases, thus removing its function. Chemical
part
is
observed
in
the
homogenizing
solution
which
contains
ethylenediaminetetr ethylenediaminetetraacetic aacetic acid (EDTA), acid (EDTA), sodium sodium dodecyl sulfate (SDS), sodium (SDS), sodium chloride (NaCl), and sodium citrate (NaC6H5O7). This solution will allow the isolation of DNA by degra degradat dating ing other other cell cell compon componen ents ts while while protec protectin ting g the DNA DNA itself itself.. In this this case, case, the EDTA serves two (2) purpose. First, it binds to the divalent metal ions (Ca
+2
, Mg+2, Mn+2)
which which could could form form salts salts with with the anion anionic ic phosp phospha hate te group groups s of the DNA. DNA. Secon Second, d, in relation to the first, it also inhibits DNases because it chelates metal ions, specifically Mg+2 or Mn+2, which which are neces necessar sary y cofac cofactor tors s of nucle nuclease ases s (Boy (Boyer, er,201 2012) 2).. SDS SDS also also performs two (2) functions, first, it solubilizes the cell membranes (emulsifying lipids and protei proteins) ns) and, and, secon second, d, being being an anion anionic ic deterg detergent ent,, it disru disrupts pts the ionic ionic inter interac actio tions ns between the positively charged histones and the negatively charged backbone of the
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Biochemistry Laboratory Formal Report DNA (Boyer,2012). The detergent then forms complexes with these lipids and proteins causing them to precipitate out of the solution (Extraction of DNA, n.d.).Sodium chloride (NaC (NaCl) l) provi provides des a shiel shieldin ding g effec effectt to nuclei nucleic c acid acid’s ’s negati negative ve phosp phospha hate te ends ends from from cation cations s (dimin (diminish ishing ing ionic ionic intera interact ction ions) s) causi causing ng the strand strands s to come come togeth together er and coalesce thus, enabling the DNA to precipitate when alcohol is added. The enzymatic part is observed in the addition of papain. Papain is a cysteine protease that denatures proteins which are clinging to strands of DNA. This allows the DNA to furthe furtherly rly uncoi uncoill making making it easier easier to spool spool,, and remov remove e impur impuriti ities es broug brought ht about about by proteins (Extraction of DNA, n.d.). The results of the experiment are shown below. Table 1. Results of the experiment Description
White, thread-like material
Absorbance
Weight (mg)
A260
A280
33.8
-0.339
-0 -0.296
Protein Ratio (mg/mL)
---
---
Nucleic acid (μg/mL) ---
II. Test for Concentration and Purity Using UV-Vis
From From the give given n data data above above,, negati negative ve absor absorpti ption on readi reading ngs s were were recor recorded ded in which which theoretically are not possible. UV-Vis spectroscopy uses the principle that conjugated pi bonds bonds absor absorbs bs light. light. From From this, this, the degr degree ee of absor absorpti ption on is calcu calculat lated ed by noting noting the difference after the light had passed through a solution. The readings from the sample is compared to a reagent blank which only contains the solvent used to dissolve the DNA. This This canc cancel els s any any unwa unwant nted ed read readin ings gs that that may may be scan scanne ned d in the the proc proces ess, s, thus thus,, produci producing ng only the DNA’s DNA’s absorban absorbance. ce. This is the reason why negative negative absorba absorbance nce values are not possible. Possible explanations to these bad readings could be: wrong or differ differen entt solve solvent nt was was place placed d in the reagent reagent blank blank (a solve solvent nt whic which h has has even even more more
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Biochemistry Laboratory Formal Report content than the DNA-SSC solution), or wrong side of the cuvette is exposed (matted side). Using a nomograph, the purity and the concentration can be quantify. Pureness of DNA isolate is based on A 260/A280 ratio. Pure DNA has a ratio of ~1.8. Before reading, DNA is dissolved in SSC solution. The reason behind this is that DNA is more stable in high saline solution. In addition, SSC is a buffer, thus, preventing any change in pH that might denature DNA and affecting the reading. Single stranded DNA (denatured) absorbs more light than those in double helix form (Sharma, A.K., 2011). III. Chemical Characterization
characterization results Table 2. Chemical characterization Chemical Test Standard Sample Dark Dark-p -pur urpl ple e solu soluti tion on Dische Test Clear Clear solut solution ion w/ yello yellow w ppt* ppt* Test for Phosphate Orange substance (crust-like) (crust-like) Murexide Test Clear, colorless solution w/ Wheeler-Johnson Test white ppt; purple litmus
DNA Hydrolyzate Clear lear,, colo colorl rles ess s solu soluti tion on Clear Clear colorl colorless ess soluti solution on Yellow substance (crust-like) (crust-like) Clear, colorless solution w/ white ppt; purple litmus
Before characterizing, DNA isolate was first hydrolyzed using an acid. Strong acids at a high high tempe tempera ratur ture e are capabl capable e of breaki breaking ng DNA DNA molec molecule ule into into its compo componen nents. ts. The The phosphate ester bonds and N-glycosidic bond between the sugar and the nitrogenous bases bases are broken broken by hydro hydrolys lysis is at this this extr extreme eme condi conditio tion. n. This, This, in turn, turn, releas releases es a mixture of 4 nitrogenous bases, deoxyribose, and phosphoric acid. First for the Dische reaction, this test can identify DNA by its sugar component, the
Scheme 1. Dische reaction
deox deoxyr yrib ibos ose. e.
Basi Basica call lly, y,
between
Dische
the
the the
reagent
reac reacti tion on or
the
diphenylamine reagent and 2-deoxyribose constitute to the development of a blue color. The reaction is dependent to the conversion of the pentose sugar to ω-hydroxylaevulinic
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Biochemistry Laboratory Formal Report aldehyde which reacts to diphenylamine to produce a blue colored compound (Scheme 1). The concentration of the DNA is directly proportional to the intensity of the color (Disc (Dische he reacti reaction on,, n.d.). n.d.). The resul resultt of the DNA DNA hydr hydroly olyza zate te in this this test test is color colorle less ss becau because se the amoun amountt DNA DNA used used is extre extremel mely y low, low, there therefor fore, e, there there is no notic noticea eable ble results. For the phosphate test, this test is basically based on the reaction of phosphate ions with
ammonium
((NH4)3PMo12O40),
molybdate
which
yields
ammon monium
phosphomolybdate
hence nce, the the yello ellow w crystal stals s (Damod amodar aran an,, 201 2011).
Again ain, the the
hydrolyzate does not form any yellow precipitate indicating a negative result. For Murexide test, this test is really for uric acid, however, uric acid is the end product of purine purine cataboli catabolism. sm. In this reaction, reaction, purines purines when when reacted reacted to concent concentrate rated d nitric nitric acid (HNO3) are oxidized to dialuric acid and alloxan which then condense to form alloxantin. This This is then then react reacted ed to potas potassiu sium m hydro hydroxid xide e (KOH) (KOH) to form form ammon ammonium ium purpu purpura rate te or murexide which is a red (pink) residue. But, purplish violet in color may also be seen due to the potassium salt (Damodaran, 2011). Once more, due to the lack of DNA for this part of the experiment, a yellow tint was just seen and not a red color. Scheme 2. Murexide formation from a purine
Lastly, Lastly, for the WheelerWheeler-John Johnson son test, pyrimidi pyrimidines nes undergo undergo bromina bromination tion upon reaction reaction with bromine water to produce produce dibromohydroxyuracil dibromohydroxyuracil which which is a yello yellow w solut solutio ion. n. The The addition of Ba(OH) 2 forms dialuric forms dialuric acid and acid and excess of it produces the purple barium salt of dialuric acid. The DNA hydrolyzate is positive for pyrimidines in this case, eventhough the observed color is white. This might be due to poor color recognition.
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Biochemistry Laboratory Formal Report Experimental methodology
Three (3) parts were involve in this experiment, namely, isolation of DNA, concentration and purity determination, and chemical characterization. First, for the isolation part, 50 mL of homogeni homogenizing zing solution solution was placed in a 250mL 250mL Erlenmeyer Erlenmeyer Flask and pre-heat pre-heated ed at 60ºC in a water bath. 25g of peeled and minced onion was made up and mixed with the homogenizing solution for 5 minutes still under 60ºC and was intermittently stirred every 2 minutes. After that, 3 pieces of papain tablets were pulvurized and added to the solution for another 10 minutes. After which, the flask was placed immediately to an ice bath and swirled occasionally. Then, the mixture was placed in a blender and blended for for 45 seco second nds. s. The The resu result ltin ing g homo homoge gena nate te was the then filt filter ered ed usin using g 4 lay layers ers of cheesecloth. The filtrate was collected with a 100mL graduated cylinder and the volume was was note noted. d. The The resi residu due, e, on the the othe otherr hand hand,, was was disc discar arde ded. d. The The filt filtra rate te was then then transferred to a 250mL beaker. After which, the beaker was placed in an ice bath in a position tilted to 45º. Cold ethanol, with a volume twice that of the homogenate, was poured slowly onto the side of the beaker to prevent disrupting the upper layer where DNA DNA will will prec precip ipit itat ate. e. It was was let let stoo stood d for for 8 minu minute tes, s, and and afte afterr whic which, h, the the DNA DNA was spooled using an improvised spooler made during the course of the preliminary part of the experiment. The spooled DNA strings was placed in a pre-weighed watch glass and weighed in an analytical balance. The resulting weight was noted. In deter determin minati ation on of DNA’s DNA’s conce concentr ntrat ation ion and purity purity,, 2.0mg 2.0mg of the DNA DNA isolat isolate e was was weighed and placed in a large test tube. After that, 6.6mL saline-sodium citrate (SSC) solution was added to the test tube (1.0mg DNA:3.3mL SSC) and mixed using a vortex. Then, using a UV-Vis, the solution was subjected to two (2) wavelengths of light (260nm
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Biochemistry Laboratory Formal Report & 280nm) at the same time and the absorbance reading was noted. The readings were plotted using a nomograph. Lastly, for the chemical characterization part, four different tests were included in this exper experime iment: nt: Disch Dische e (Test (Test for deox deoxyr yribo ibose se), ), Murex Murexide ide (Test (Test for purine purines), s), Wheel WheelererJohnson (Test for pyrimidines), and phosphate test. Before these tests, the DNA isolate was first hydrolyzed with an acid. The excess DNA isolate was transferred first in a large test tube and 1mL of 1M HCl (hydrochloric acid) was added to it. Then, the test tube was covered with a marble and was heated in a boiling water bath for 1 hour with occasional shaking. After that, it was cooled to room temperature and, then, added with 2.5mL distilled water. It was then neutralized with 1M NaOH (sodium hydroxide) and filtered. The resulting filtrate was diluted with a distilled water to make 4mL of a solution. Then, the DNA hydrolyzate was ready for chemical characterization. First, for Dische reaction, 1.5mL of diphenylamine reagent was added to 1.0mL of the DNA hydrolyzate. The solution was heated in a boiling water bath for exactly 10 minutes. Then, it was cooled immediately and the results were observed. For the test for phosphate, 1mL of concentrated H 2SO4 (hydrochloric acid) was added to 1mL of DNA hydrolyzate. Then, 0.5mL of concentrated HNO 3 (nitric acid) and 1mL of distilled water were added. After that, it was heated in a boiling water bath for 5 minutes. The solution solution was allowed allowed to cool and, after after cooling cooling,, 1mL of ammonium ammonium molybdenate molybdenate solution was added to it. The solution was mixed well and diluted with 10mL of water. It was let stood for 10 minutes. The solution was then observed (if there are precipitate formed and color change).
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Biochemistry Laboratory Formal Report For the Murexide test, a small amount of the DNA hydrolyzate (about 3-5 drops) was placed in a small evaporating dish. Then, a few drops of concentrated nitric acid (HNO 3) was was adde added d to it. it. It was was then then evap evapor orat ated ed to dryn drynes ess s in a wate waterr bath bath.. The The resu result ltin ing g residue was moistened with 10% KOH (potassium hydroxide). The change in color upon the addition of KOH was noted. Then, a few drops of water were added and was heated (warmed) again. Then, the solution’s color was observed. After which, the solution is evaporated again and the resulting residue was observed for its color. Lastly Lastly,, for the Wheele Wheeler-J r-Joh ohnso nson n test, test, 0.5mL 0.5mL of DNA DNA hydr hydroly olyza zate te was was treate treated d with with excess bromine water (Br 2•H2O) until the solution turned yellow. To remove the excess bromine water, the solution was boiled using a water bath until it became colorless or light yellow. After which, barium hydroxide (Ba(OH) 2) was added in excess. Then, the color was noted. The same treatment was done to the each standard affiliated to each test. The results from the standard was also noted and compared to that of the sample.
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Biochemistry Laboratory Formal Report References
For journal resources Wheeler, H.L., & Johnson, T.B. (1907). Researches on pyrimidines: On color test for uracil and cytosin. J. Biol. Chem. , 3, 183-189. For book references Boyer, R.F. (2012). Biochemistry laboratory : modern theory and techniques (2nd Ed.). Upper Saddle River, N.J.: Pearson Prentice Hall. Campbell, M.K., & Farell, S.O. (2012). Biochemistry (7 (7th Ed.). Ed.). USA: Brooks/Cole, Brooks/Cole, Cengage Learning Damodaran, G.K. (2011). Practical biochemistry. biochemistry. New New Delhi: Jaypee Brothers Medical Publishers (P) Ltd. Sharma, A.K. (2011). Biochemistry of nucleic acid. New Delhi: Random Publications. For web resources Dische reaction. (n.d.) Retrieved from: http://product.lookchem.com/item/251/diphenylamine-test.html Extraction of DNA from onion cells. (n.d.) Retrieved from: http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/cpmcnet.columbia.edu/d ept/physio/tchrplan/oniondna.html
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