ISOLATION, HYDROLYSIS, AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS FROM Allium Cepa Martin R. Hernandez Elina Andrea S.

Lantion Jhoe Cynder P. Legaspi James Timothy A. Liwag Alisson Mae T. Mangabat Mikee D.A. Melad !"Pharma#y $io#hemistry Laboratory ABSTRACT D%A is present in all organi# materials &o'nd on the &a#e o& the planet. (t is the geneti#) hereditary material &or all organi# material. (t is able to reprod'#e and takes &orm as a do'ble" heli* #on&ormation. +e isolated D%A &rom white onion ,Allium cepa)) #hara#terized its n'#lei# a#id #omponent 'sing its a#id hydrolysate. -e.eral /'alitati.e tests are per&ormed on this n'#lei# a#id isolate to &'rther #lassi&y its #ontents and as #on&irmation tests as well. INTRODUCTION The in&ormation in D%A is stored as a #ode made 'p o& &o'r #hemi#al bases0 adenine ,A1) g'anine ,21) #ytosine ,C1) and thymine ,T1. D%A bases pair 'p with ea#h other) A with T and C with 2) to &orm 'nits #alled base pairs. 3a#h base is also atta#hed to a s'gar mole#'le and a phosphate mole#'le. Together) a base) s'gar) and phosphate are #alled a n'#leotide. %'#leotides are arranged in two long strands that &orm a spiral #alled a do'ble heli*. The str'#t're o& the do'ble heli* is somewhat like a ladder) with the base pairs &orming the ladder4s r'ngs and the s'gar and phosphate mole#'les &orming the .erti#al sidepie#es o& the ladder.

Fig. 2 Chemical Structures of DNA Bases

E !ERIMENTAL I. Material" A. DNA Isolatio 5ellow onion 67 -D- 8 9.:6 M %aCl 8 9.:6 -odi'm Citrate ; homogenizing sol'tion Papain or meat tenderizer (#e"#old <67 3thanol B. Aci! H"!rol"sis :M HCl :M =>H 2la#ial A#eti# A#id ?HoAC@ C. #ualitati$e A al"sis :. Te"t #or deo$%ri&o"e 'Di"()e* Diphenylamine reagent -td. sol'tion

Fig. 1 Double Helix structure of DNA

. Te"t #or !)o"+)ate Con#. H ->A Con#. H%>B :97 ,%HA1 Mo>A B. Te"t #or !,rine" $romine water $a,>H1

stirringEmi*ing. Co.er the t'bes with marbles Cool test t'bes 'nder r'nning water and add :M =>H to nea'tralize >btain a pH o& A"C with gla#ial a#eti# a#id ?make 'se o& pH paper to be able to dete#t pH@ Centri&'ge and de#antF to be s'bGe#ted to #hara#terization.

II. !ro(ed,re A. D%A (solation Heat 69 mL o& homogenizing sol'tion in a : 6"mL 3rlenmeyer &lask in a water bath 'ntil it rea#hes C9 C. +eigh o't 6g o& min#ed onion Add the min#ed onion to the pre.io'sly heated homogenizing sol'tion. -tir and let it sit in the water bath &or 6 mins. Add AmL o& meat tenderizer sol'tion and keep it in the C9C water bath &or :9 mins. (mmedieately pla#e the &lask in an i#e bath &or 6 mins. Allow the sol'tion to #ool e.enly as not to impede the digestion o& deo*yribon'#leases. D'i#kly b't sa&ely po'r o't the mi*t're into a blender and allow to homogenize &or A6 se#onds. Cool on i#e. !ilter yield in A layers o& #heese#loth into a 69 mL beaker. Add :6" 9 ml o& i#e #old ethanol immediately) by 'sing a pipette and allowing it to drip on the side o& the test t'be walls. Let stand &or B"6 mins witho't dist'rbing it. -pool the inta#t D%A by 'sing a makeshi&t hook o't o& a bent pipette. Trans&er isolated D%A and re#onstit'te with T3 b'&&er system. $. A#id Hydrolysis Mi* D%A -ample with :M HCl Heat sol'tion at :99 C &or : ho'r with #ontin'o's or #onstant

C. D'alitati.e Analysys -. Te"t #or +,rine" 'Di"()e te"t* Add B.6 mL o& diphenylamine reagent to :.6 mL o& hydrolyzed D%A -ol'tion. Repeat pro#ed're &or 9.6 ml o& standard deo*yribose sol'tion Pla#e both t'bes in a water bath ,boiling1 &or :9 mins. Cool immediately and obser.e res'lts. .. Te"t #or !)o"+)ate" Add : ml #on#entrated H ->A to : ml o& n'#lei# a#id isolate sol'tion. ,Repeat pro#ed're to phosphate standard sol'tion1 Heat both t'bes while shaking &re/'ently 'ntil the #ontents t'rn brown. Cool both mi*t'res and add 9.6 ml #on#. H%>B. Heat 'ntil white &'mes are obtained. (& the li/'id is still not #lear) repeat step B. Add : ml water to the #olorless li/'id and heat &or 6 mins. in a boiling water bath.. Cool and add :ml :97 ,%HA1 Mo>A sol'tion. Mi* well and dil'te to :9mL with water) and let stand &or 6 mins. obser.e the #olor o& the pre#ipitate &ormed. /. Te"t #or !,rine" 'M,re$ide te"t* Pla#e 6":9 drops o& n'#lei# a#id sol'tion into an e.aporating dish) and add a &ew drops o& #on#. H%>B. Repeat pro#ed're &or standard p'rine sol4n.

3.aporate in a water bath 'ntil dry. Moisten the resid'es &ormed with :97 =>H and heat &'rther. %ote #olor #hanges a&ter addition o& =>H and s'bGe#tion to heat. Add a &ew drops o& water and warm) take note o& the sol'tion4s #olor. 3.aporate and take note o& the #olor o& the resid'e.

http0EEwww.genome.go.E 66 9HH9 *aborator" +a ual i (e eral Biochemistr", 5srael M.) et. al. , 99<1

0. Te"t #or !%ri1idine" '2)eeler3 4o)n"on* Treat 9.6 mL n'#lei# a#id sol'tion) and standard sol'tion with an e*#ess o& bromine water) 'ntil the sol'tion t'rns yellow. Remo.e e*#ess water by boiling the sol'tion 'ntil yo' yield a light yellow to #olorless sol'tion. Add e*#ess $a,>H1 sol'tion and test with litm's paper) take note o& sol'tion4s #olor. RESULTS AND DISCUSSION C)e1i(al Te"t Test &or p'rines Test &or ribose Std. Sol,tion DNA #ro1 Onion 3merald green Red" orange resid'e P'rple sol'tion 5ellow ppt. '5* re",lt 3merald green Red" orange resid'e P'rple sol'tion 5ellow ppt.

3merald green Red" orange resid'e Test &or P'rple pyrimidines resid'e Test &or 5ellow phosphate ppt.

%able 1. Chemical Characteri&atio

Res'lts show that the isolated D%A &rom sample Allium cepa is positi.e &or all tests. P'rines) ribose) pyrimidines and phosphate are all present in the isolated D%A. REFERENCES DNA ' (e etic Structure retrie.ed &rom http0EEghr.nlm.nih.go.EhandbookEbasi#sE dna Deox"ribo uclease) Retrie.ed &rom