1320 PRINCIPLES OF PHYSICAL CHEMISTRY The numbering of the postions in the rings has been established by IUPAC convention. The major pyrimidines found in‘DNA are shymine and cosine ; ix RNA, they are uracil and cytosine ; a ; i I i Neca, yw yon I i L I 1 ea} aon y W ¥ oo eo oo ee enor p 5 P } io ir SN ae SA Nu el wl SAY ANY? pe = [DNA molecules comprise more thn 10¥ aucleoies. They contain adenine, thymine, guanine and cytosine asthe bases, and the genetic information is encoded within the nucleotide sequence which is precisely defined over the entire length of the molecule. One of the simplest methods for determining the nucleotide sequence of DNA makes use of the enzyme, DNA polymerase,which catalyzes the synthesis of DNA. . DNA is a duplex molecule in which two polynuclenide chains (or strands) are linked t0 one another through specific base pairing (Fig, 22). Adenine in one strand is paired to thymine and i i ay yf f Fi. 2, Bas psig ia DNA guanine is paired 10 cytosine. The two chains are said 10 be complementary. This was one of the essemial features of the Watson-Crik meade ofthe structure of DNA. Hydrogen bonds ae formed between the opposing bases within par. Inthe strvtue proposed by Watson and Crick, A~~-T and G.-C base pars are roughly plantr, with H bonds (ted lines) a8 shown in Fig. 22. It may be roted that two H bonds are formed in an A:T pair and three in a G:C pait. The base pairs are stacked on top of one another, the plane ofthe base pais being perpendicular to the length of the MACROMOLECULES * 1321 duplex, Tiss stowndagrmaialy in te ladderpe acne (Fg 23). Sent end This motel for DNA. inorporning , on ae pig been compensa | ey At eg ‘and consistent with X-ray diffraction data} 5 s vy ts proposed by J.D. Watson and FC. | |g co A ick e195, base ote ster was 7 Ss, the twisting of the two strands around one Po another to give arightchanded tlix ie || S&T — Tome — 8 doable bel) and fo achieve «sete r fedssent wih the dla avalable at that oe Gn fe, Twat oceatry "ent Ie 7 ; ‘complementary chains in opposite directions vr ay cares (ig 29, Dice poot for his oppotte | S—— So: Parity in chin Gieton was stieved a ¥ Se oo Je ae eit interes in tnovng et. Sig fa pi ith DNA de Te it tow the twisting of the two srands © | ES recap Wtoispeween te seep wipe fhe a fell onic oe sy of jouer the overall strctre of DNA, In fat oe of the mos significant tects of twisting into abel if bring the stacked pie very cose wo one footer. The double helix can exist in several forms, Inthe sealed B form the heli (Fi. 28), this diane, whieh i called the rise, is O34 tim, Consoquenty, water is exchided fom what FS now a hytobobc coe; th charged popes | = are onthe surface. The hydeopobi erations Within the core contribute, long with te Hk Boods tetween the base pis, o the overll sabi of the tll, Ieshoud sla be ated hat tere is one complete twist ofthe heli evety 10, tase pais or 3-4 nm. This dances ceferred to a the ptch ofthe helix, The surfce of he helix shows alternating major an mina grooves wich follow: the twist ofthe dole sanded molecale slong is length. The major groove i tow foown to accommodate ineracios with proteins that recognize and bind to specific cleat sequences Supereailed DNA. Soperotng represent & | 4 ing of the DNA. dole tli opon et | ¢™*, cccars in cralar DNAt and in DNA tht ae topologically constrained by being complexed ote, Ifa eirelar DNA melee in related enformation (no sopecoilig) is broken sess bath strands and one or more adios ight anda tial une are inserted before refining the ends, the molecule woul twit on itl form a positive supercoil If on he oder band, the bei is unwound (given left handed tus) before rejoining, the result i negative superol. These Minor prove 2400] ior grove Fig. 24. Diagram of te DNA double tlic (esl for,1322 PRINCIPLES OF PHYSICAL CHEMISTRY forms, called topomers, ae shown in Fig 25. Fach supercoil depicted hete contains single supertwist, The number of supertwists in a ‘molecule can be very large, Supercoiled DNA is readily s famverted into its relaxed form by the introduction of a break (or ‘ob beeen ajacet nls AG ‘of the two strands ; this | Negative superéoil Positive supereoit ae Jonger topologically | pig ss, Depiction of nepaive ad positive sepercails ina supercoiled DIA. Denaturation of DNA. The double heli ia relatvely stiff and elongated molecule. Conequent 2 soinion af DNA has high voy, If ah 9 slutta I heated te 33°C. econ of fason dops markedly, reesng 2 claps of te Cxtieelea Sowcre. Ts Kaown Aeaturaton and accompanied by sepation of be caples toi single sane whch ely ble Deranraioa and remsuaton poi valbi oman cn inpoan poperes ofthe DNA oie om ri Sources Desatraton slo poids the bs fey [Hesse and sensitive spponthes fo te ienifeaton of seen nfo DNAs ANY of praous sgifesns rte rapid eveopmes in molecular genetics. While densraon can be dete iy th changes in vscosiyy 2 much tae conteien ay 2 seret it by ave (UV) sbexpon mess The dfecnee in the UY absorption spe of tive (Goble eica) and dened (sg sanded) foms of DNA fs showa ia Fig. 26. A he woe ath Reet entormain Aborbance B39 3 ‘of maximum absorption (260 nm), absorptica le | acne Stand DNAs approumately 40 pr ces higher han | PE Abin pee dna a that by double-stranded DNA.. This is teered to asthe hyperchromic effect and results from the westackng of the base pars inthe helix. The DNA ei esky ods een ni is wel 5 by hyp foes etmesn stacked tase pie, pooner nee Pw wel a Reagents that reduce the H bonding oy ani eee‘. poly oe Strounding eden Such.‘ ‘oman, wil ese dnauron, Exeones of i which tow te beer care ets efor & Preomncocis ‘Relnive stvortance at 260 om ‘Thus, DNA at pH 10 shows absorption . ‘at 260 nm which is 40 per cent “ Higher than that ofthe native form. | we If the temperaure of a solution of DNA is increased eradvally, the a change tothe denatured form can be | 10 monitored by change in absorbance at eee Tempers gg) 260 nm. Typical results for several ‘ypes of DNA are shown in Fig, 27, Fig. 7. Maing cares deren ypes of DNA, MACROMOLECULES 1323 ‘The curves are referred to 25 melting curves because the region over which the absorbance increases reflects the collapse (or melting) of the highly organized, semicrystalline sci of doube-hetical DNA. ‘Tee temperature a which 50 per ceu melting has occured is alld Uae malting temperature ot Ty. ‘he Ty at neural pH is depeniet on the ‘oni atength ofthe medium. The cures shown Fig. 27 are thote obtained at anionic stength of jst above 0-15 Ifthe ionic sengi is reduced by 90 per cent, all Ty valuts ate lowered by about 20°C. This results from the additonal negative charge and consequently greater electrostatic repulsion (which helpe disruption of the els) tia the DNA structure a the lower one svengh The DNAS fom differen sources have direst ry ves. This is because the DNAS have diferent atouais of G:C and A:T bate pls andthe fomer tonfer greater stability on the belx trough the resece of tice H Bonds per base pal than vo. _ Tins, te higher the G-+C content, the higher the Ty, Tbe vale of Ty under standard conditions can te wed, therefore, to obtain an estimate of the (G+C) conteat of an unknown DNA. This is evident fiom Fig. 28 which shows a plot of Ty versus | P€%- Meiag semper of DIVA «= fncton (GC) cone of number of DNAS. Renaturation of DNA. The complementary sands of DNA, separated by heat, spontaneously reasoiate when the temperature is lowered below te Tq. This renataration is also refered © a3 Tmnealag. The rac of annealing depends onthe eomceataion of complementary sequences, Vira DNA has a snaler variety of sequences than does bacterial DNA. ; his reflects the higher level of ened complexity in bacteria. Thus, for viel and bacterial DNA fragments of the same average Sice and at the same molar concentration, there-woul be 2 higher concentration of complementary Sequences in the fomer. Vial DNA, therefore, woul rentrate faster than bacterial DNA ‘The Polymerase Chain Reaction (PCR). In order obainsuiient quantities ofa partcwar DNA sample ta enable, determination of is properies, we make use of the polymerase chain reaction (PCR) which i, inded, a pe of chain reaction. PCR provides a way to generate a vast umber of double-stranded copies of a specific DNA sequence. ‘The polymerase chain reaction uses : (1) a thermostable DNA’ poyinerase, 2) a DNA template which isto be amplified, (3) two primers, each pally of 20 nucleotides, which anneal to distinct farts onthe complementary strands ofthe target and serve as sts for commencing DNA polymerase ‘reaction, (4) a solution containing deoxynucleoside triphosphates, Mg?* salts and pH buffer. PCR uses DNA polymerase to maie complementary copies of DNA corresponding to the region of inerest.To direct the enzyme to the. corect sequence, PCR relles on primers which anneal to fomplemeatary sequences in target singlestanded DNA. DNA polymerase cannot stat. DNA ‘jtiesis de novo and can extend the chin only fom the amealed primers. An excess of these imers & provided in the reaction minute to allow sufceat mattial for generating ampli Poteet. PCR reactions inde the ejecal we of hgh temperatures which can lead 10 the {eatuaon of ermal enzymes. ‘Widhout going ino the dts ofthis very imporaat reaction, we mention here that for PCR, there ae only thee steps invoviag DNA, These ae (2 double-stranded DNA denaturation, (2) DNA ‘annealing and (3) DNA extension, Eacl step is intially conducted at a different temperature in a Programmable machine called thermal cycler. A sep’ inthe chain reaction typically takes less Mol percent (G40)