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An actual sequencing gel is shown in Fig. 12-35.+•HimdATP5'Primerstrand3 ' A C G G C T A C TTemplatestrand00000000(a)O"O"O"IoIoHHddNTP Analog(b)3 I H l l H i CTAAGCTCGACTTemplatedCTP, dGTP, dATP, dTTPddATPGATTCGAGCTGddAGATTCGddAGddA- ddCTPI GATTCGAGddCI GATTddC-ddGTPGATTCGAGCTddGGATTCGAddGGATTCddGddG•ddTTPI GATTCGAGCddTI GATddTI GAddT\G121110987654321Autoradiogram ofelectrophoresisgel(c)3'AGTCGAGCTTAGSequence ofcomplementarystrandChapter 12 Nucleotides and Nucleic Acids36).
The Sanger method (Fig. 12-36) is in more widespread use because it has proven to be technically easier. It involves the enzymaticsynthesis of a DNA strand complementary to the strand to be analyzed.DNA sequencing is now automated, using a variation of Sanger'ssequencing method in which the primer used for each reaction is labeled with a differently colored fluorescent tag (Fig. 12-37).
This technology allows sequences of thousands of nucleotides to be obtained in afew hours, and very large DNA-sequencing projects are being contemplated. The most ambitious of these, now underway, is the HumanGenome Initiative, in which all of the 3 billion base pairs of DNA in ahuman cell will be sequenced.Colored^dye labelPrimer/ 3'349- Template ofunknown sequenceDNA polymerase,4 dNTPs, one ddNTP (C)o• CDenatureFigure 12-37 A prototype strategy for automatingDNA sequencing reactions. The short oligonucleotides used as a primer for DNA synthesis in theSanger method can be linked to a fluorescent molecule that gives the DNA strand a color.
If each nucleotide is assigned a different color, the nucleotideon the end of each fragment can be identified bycolor. The dideoxy method is used with a differentddNTP added to each of the four tubes according tothe color assignments. The resulting colored DNAfragments are mixed and then separated by size ina single electrophoretic gel lane. The fragments of agiven length migrate through the gel in a peak,and the color associated with each successive peakis detected using a laser beam. The DNA sequenceis read by determining the sequence of colors in thepeaks as they pass the detector, and this information is fed directly to a computer.oooooiA\AXXJLr_J_JJUUUL_JThesequenceinformationis fed to acomputer.DNAmigrationDye-labeledsegments of DNA,copied fromtemplate withunknown sequenceCFour separatereaction mixturesas shown abovefor C (primers foreach nucleotideare attached todye-label ofdifferent color)The products of thefour reactions aremixed and all dyelabeled segments areapplied to a singlelane of the gel.ATAGCTGTTTCTGCAGTGCCDetectorLaser beam350Part II Structure and CatalysisFigure 12-38 Automated synthesis of DNA is conceptually similar to the solid-state synthesis of polypeptides.
The desired oligonucleotide is built up ona solid support (silica) one nucleotide at a time in arepeated series of chemical reactions with suitablyprotected nucleotide precursors. (T) The first nucleotide (which will be the 3' end) is attached to thesilica support at the 3' hydroxyl (through a linkinggroup, R), and is protected at the 5' hydroxyl withan acid-labile protecting group (dimethoxytrityl,DMT). The reactive groups on all bases are alsochemically blocked. (2) The protecting DMT groupis removed by washing the column with acid (theDMT group is colored, so this reaction can be followed spectrophotometrically).
(3) The next nucleotide is activated and reacted with the bound nucleotide to form a 5'-3' linkage, which in (4) isoxidized with iodine to produce a phosphotriesterlinkage. (One of the phosphate oxygens is methylated.) Reactions (2) through (4) are repeated untilall nucleotides are added. At each step, excess nucleotide is removed before addition of the next nucleotide. In (§) and (6) the remaining blockinggroups on the bases and the methyl groups on thephosphates are removed, and in (7) the oligonucleotide is separated from the solid support and purified. The chemistry of RNA synthesis has laggedfar behind the procedures for DNA synthesis because of difficulties in protecting the 2' hydroxylof ribose without adverse effects on the reactivityof the 3' hydroxyl.DMT-0Nucleotideprotectedat 5' hydroxylHH^13' Nucleotideattached tosilica supportDMT-0DMTProtectinggroup removedThe Chemical Synthesis of DNA Has Been AutomatedAnother technology that has paved the way for many biochemical advances is the chemical synthesis of oligonucleotides with any chosensequence.
The chemical methods for synthesizing nucleic acids weredeveloped primarily by H. Gobind Khorana in the 1970s. Refinementand automation of these methods has made it possible to synthesizeDNA strands rapidly and accurately. The synthesis is carried out withthe growing strand attached to a solid support (Fig. 12-38), using principles similar to those used by Merrifield in peptide synthesis (see Box5-2).
The efficiency of each addition step is very high, allowing theroutine laboratory synthesis of polymers of 70 or 80 nucleotides. Insome laboratories much longer strands are synthesized. The availability of relatively inexpensive DNA polymers with predesigned sequences is having a powerful impact on all areas of biochemistry(Chapter 28).Other Functions of NucleotidesIn addition to their roles as the subunits of nucleic acids, nucleotideshave a variety of other functions in every cell: as energy carriers, components of enzyme cofactors, and chemical messengers.Nucleotides Carry Chemical Energy in CellsNucleotides may have one, two, or three phosphate groups covalentlylinked at the 5' hydroxyl of ribose.
These are referred to as nucleosidemono-, di-, and triphosphates, respectively (Fig. 12-39). Starting fromChapter 12 Nucleotides and Nucleic AcidsNucleotideactivatedat 3' hydroxylDMT-0DMT-0351DMT-01CHBase22\HHk H•••HOCH3O—PCH3O-P=OCH 3 O-P+(CH 3 ) 2 CH-N -CH(CH 3 ) 2H0|CH2OnHNext nucleotideadded/HOMBOxidationto form triesterHRISiwH0i1RV(CH 3 ) 2 CH-N-CH(CH 3 ) 2H.0HHH\\^H0H'\~i1SiRepeat steps2 to 4 until all residues are added5 Remove blocking groups from bases6 Remove methyl groups from phosphates7 Cleave chain from silica support•3'5'Oligonucleotide chainthe ribose, the three phosphates are generally labeled a, f}, and y.
Nucleoside triphosphates are used as a source of chemical energy to drivea wide variety of biochemical reactions. ATP is by far the most widelyused, but UTP, GTP, and CTP are used in specific reactions. Nucleoside triphosphates also serve as the activated precursors of DNA andRNA synthesis, as will be described in Chapters 24 and 25.y(30I0"IFigure 12-39 General structure of nucleoside 5'mono-, 5'-di-, and 5'-triphosphates (NMPs, NDPs,and NTPs) and their standard abbreviations. In thedeoxyribonucleoside phosphates (dNMPs, dNDPs,and dNTPs) the pentose is 2'-deoxy-D-ribose.a0~I"0—P—0—P—O—P—O—CH 200oHH\OHNMPNDPNTPBaseHI/HOHAbbreviations of ribonucleoside5'-phosphatesAbbreviations of deoxyribonucleoside5'-phosphatesBaseMono-Di-Tri-BaseMono-Di-Tri-AdenineAMPADPATPAdeninedAMPdADPdATPGuanineGMPGDPGTPGuaninedGMPdGDPdGTPCytosineCMPCDPCTPCytosinedCMPdCDPdCTPUracilUMPUDPUTPThyminedTMPdTDPdTTP352Part II Structure and CatalysisThe hydrolysis of ATP and the other nucleoside triphosphates is anenergy-yielding reaction because of the chemistry of the triphosphatestructure.
The bond between the ribose and the a phosphate is an esterlinkage. The a-/3 and /3-y linkages are phosphoric acid anhydrides(Fig. 12-40). Hydrolysis of the ester linkage yields about 14 kJ/mol,whereas hydrolysis of each of the anhydride bonds yields about 30kJ/mol. In biosynthesis, ATP hydrolysis often drives less favorablemetabolic reactions (i.e., those with AG°' > 0). When coupled to a reaction with a positive free-energy change, ATP hydrolysis shifts the equilibrium of the overall process to favor product formation (recall therelationship between equilibrium and free-energy change described inChapter 8).Figure 12-40 The phosphate ester and phosphoricacid anhydride bonds of ATP. Hydrolysis of an anhydride bond yields more energy than hydrolysis ofthe ester.
A carbon anhydride and ester are shownfor comparison.Ester"1oI0-P—0-P—O—P-O-CH2IIIIIII£)OjOAnhydride LIAnhydrideHHH\ATPCH3 -C—0—C-CHg0Adenine0Acetic anhydride,a carbon anhydride/HOH OHCH3 - C - 0 - C H 30Methyl acetate,a carbon esterIt is appropriate to ask why ATP serves as the primary carrier ofenergy in the cell. The chemical energy potential of pyrophosphate(—33 kJ/mol), a much simpler molecule, is almost identical to that ofATP because pyrophosphate also contains a phosphoric acid anhydride. Pyrophosphate would be so much easier to synthesize than ATPthat the selection of ATP at first seems to contradict evolutionary logic.The explanation can be found in the fundamental energetic principles governing every chemical reaction. In promoting chemically unfavorable reactions such as those in many biosynthetic processes, the cellmust deal with both the equilibrium and the rate of the reaction.
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