《生物化学原理》（英文版）chapter 8 NUCLEOTIDES AND NUCLEIC ACIDS

8.3 Nucleic Acid Chemistry 293 duplexes, in which segments of a mouse DNA strand in this chapter. Hybridization techniques can be varied form base-paired regions with segments of a human dna to detect a specific RNA rather than DNA. The isolation strand(Fig. 8-32). This reflects a common evolutionary and identification of specific genes and RNAs rely on heritage: different organisms generally have some pro- these hybridization techniques. Applications of this teins and RNAs with similar functions and, often, simi- technology make possible the identification of an indi lar structures. In many cases, the DNAs encoding these vidual on the basis of a single hair left at the scene of a proteins and RNAs have similar sequences. The closer crime or the prediction of the onset of a disease decades the evolutionary relationship between two species, the before symptoms appear(see Box 9-1) more extensively their DNAs will hybridize. For exam- ple, human DNA hybridizes much more extensively with Nucleotides and Nucleic Acids Undergo mouse DNA than with DNA from yeast. The hybridization of DNA strands from different Nonenzymatic Transformations ources forms the basis for a powerful set of techniques Purines and pyrimidines, along with the nucleotides of sential to the practice of modern molecular genetics. which they are a part, undergo a number of spontaneous A specific DNA sequence or gene can be detected in the alterations in their covalent structure. The rate of these presence of many other sequences, if one already has reactions is generally very slow, but they are physio- an appropriate complementary DNA strand (usually la- logically significant because of the cell s very low toler beled in some way)to hybridize with it( Chapter 9). The ance for alterations in its genetic information. Alter- complementary DNA can be from a different species or ations in DNA structure that produce permanent from the same species, or it can be synthesized chemi- changes in the genetic information encoded therein are cally in the laboratory using techniques described later called mutations, and much evidence suggests an inti. mate link between the accumulation of mutations in an individual organism and the processes of aging and carcinogenesis Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups(deamination)(Fig g 8-33a). For example, under typical cellular conditions, deamination of cytosine(in DNA)to uracil occurs in about one of cytidine residues in 24 This corresponds to about 100 spontaneous events per day, on average, in a mammalian cell. Deamination of adenine and guanine occurs at about 1/100th this rate Sample 2 The slow cytosine deamination reaction seems in- Mⅸ nocuous enough, but is almost certainly the reason why DNA contains thymine rather than uracil. The produc of cytosine deamination(uracil) is readily recognized foreign in DNA and is removed by a repair system ( Chapter 25). If DNA normally contained uracil, recog. nition of uracils resulting from cytosine deamination would be more difficult, and unrepaired uracils woule lead to permanent sequence changes as they were Duplex df paired with adenines during replication. Cytosine deam- ination would gradually lead to a decrease in G=C base whrid-rd 加a pairs and an increase in a-U base pairs in the dna of all cells. Over the millennia, cytosine deamination could eliminate G=C base pairs and the genetic code that de- IGURE 8-32 DNA hybridization. Two DNA samples to be compared pends on them. Establishing thymine as one of the four are completely denatured by heating. When the two solutions are bases in DNA may well have been one of the crucial ixed and slowly cooled, DNA strands of each sample associate with turning points in evolution, making the long-term stor their normal complementary partner and anneal to form duplexes If age of genetic information possible the two DNAs have significant sequence similarity, they also tend to Another important reaction in deoxyribonu form partial duplexes or hybrids with each other. the greater the se. cleotides is the hydrolysis of the N-B-glycosyl bond be quence similarity between the two DNAS, the greater the number of tween the base and the pentose(Fig.8-33b). This oc hybrids formed. Hybrid formation can be measured in several ways. curs at a higher rate for purines than for pyrimidines One of the DNAs is usually labeled with a radioactive isotope to sim. As many as one in 10 purines(10,000 per mammalian plify the measurements. cell)are lost from DNA every 24 hours under typical

duplexes, in which segments of a mouse DNA strand form base-paired regions with segments of a human DNA strand (Fig. 8–32). This reflects a common evolutionary heritage; different organisms generally have some pro￾teins and RNAs with similar functions and, often, simi￾lar structures. In many cases, the DNAs encoding these proteins and RNAs have similar sequences. The closer the evolutionary relationship between two species, the more extensively their DNAs will hybridize. For exam￾ple, human DNA hybridizes much more extensively with mouse DNA than with DNA from yeast. The hybridization of DNA strands from different sources forms the basis for a powerful set of techniques essential to the practice of modern molecular genetics. A specific DNA sequence or gene can be detected in the presence of many other sequences, if one already has an appropriate complementary DNA strand (usually la￾beled in some way) to hybridize with it (Chapter 9). The complementary DNA can be from a different species or from the same species, or it can be synthesized chemi￾cally in the laboratory using techniques described later in this chapter. Hybridization techniques can be varied to detect a specific RNA rather than DNA. The isolation and identification of specific genes and RNAs rely on these hybridization techniques. Applications of this technology make possible the identification of an indi￾vidual on the basis of a single hair left at the scene of a crime or the prediction of the onset of a disease decades before symptoms appear (see Box 9–1). Nucleotides and Nucleic Acids Undergo Nonenzymatic Transformations Purines and pyrimidines, along with the nucleotides of which they are a part, undergo a number of spontaneous alterations in their covalent structure. The rate of these reactions is generally very slow, but they are physio￾logically significant because of the cell’s very low toler￾ance for alterations in its genetic information. Alter￾ations in DNA structure that produce permanent changes in the genetic information encoded therein are called mutations, and much evidence suggests an inti￾mate link between the accumulation of mutations in an individual organism and the processes of aging and carcinogenesis. Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups (deamination) (Fig. 8–33a). For example, under typical cellular conditions, deamination of cytosine (in DNA) to uracil occurs in about one of every 107 cytidine residues in 24 hours. This corresponds to about 100 spontaneous events per day, on average, in a mammalian cell. Deamination of adenine and guanine occurs at about 1/100th this rate. The slow cytosine deamination reaction seems in￾nocuous enough, but is almost certainly the reason why DNA contains thymine rather than uracil. The product of cytosine deamination (uracil) is readily recognized as foreign in DNA and is removed by a repair system (Chapter 25). If DNA normally contained uracil, recog￾nition of uracils resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent sequence changes as they were paired with adenines during replication. Cytosine deam￾ination would gradually lead to a decrease in GqC base pairs and an increase in AUU base pairs in the DNA of all cells. Over the millennia, cytosine deamination could eliminate GqC base pairs and the genetic code that de￾pends on them. Establishing thymine as one of the four bases in DNA may well have been one of the crucial turning points in evolution, making the long-term stor￾age of genetic information possible. Another important reaction in deoxyribonu￾cleotides is the hydrolysis of the N-
-glycosyl bond be￾tween the base and the pentose (Fig. 8–33b). This oc￾curs at a higher rate for purines than for pyrimidines. As many as one in 105 purines (10,000 per mammalian cell) are lost from DNA every 24 hours under typical 8.3 Nucleic Acid Chemistry 293 FIGURE 8–32 DNA hybridization. Two DNA samples to be compared are completely denatured by heating. When the two solutions are mixed and slowly cooled, DNA strands of each sample associate with their normal complementary partner and anneal to form duplexes. If the two DNAs have significant sequence similarity, they also tend to form partial duplexes or hybrids with each other: the greater the se￾quence similarity between the two DNAs, the greater the number of hybrids formed. Hybrid formation can be measured in several ways. One of the DNAs is usually labeled with a radioactive isotope to sim￾plify the measurements

Chapter8Nucleotides and Nucleic Acids Uracil 0-CH 0 Guanosine residue NH2 CH3 CH3 5-Methylcytosine Th NH2 HON N % Canine Adenine Hypoxanthin Aparima rsidue b)Depurination FIGURE 8-33 Some well-characterized nonenzymatic reactions of nucleotides. (a) Deamination reactions. Only the base is shown. (b)Depurination, in which a purine is lost by hydrolysis of the N-B Guanine Xanthine glycosyl bond. The deoxyribose remaining after depurination is readily converted from the B-furanose to the aldehyde form (see Fig. 8-3).Fur ther nonenzymatic reactions are illustrated in Figures 8-34 and 8-35 cellular conditions. Depurination of ribonucleotides and human skin cells. We are subject to a constant field of RNA is much slower and generally is not considered ionizing radiation in the form of cosmic rays, which can physiologically significant. In the test tube, loss of penetrate deep into the earth, as well as radiation emit purines can be accelerated by dilute acid Incubation of ted from radioactive elements, such as radium, pluto DNA at ph 3 causes selective removal of the purine nium, uranium, radon, C, and H X rays used in med bases, resulting in a derivative called apurinic acid. cal and dental examinations and in radiation therapy of Other reactions are promoted by radiation. UV light cancer and other diseases are another form of ionizing induces the condensation of two ethylene groups to radiation. It is estimated that UV and ionizing radiations form a cyclobutane ring. In the cell, the same reaction are responsible for about 10% of all DNA damage caused between adjacent pyrimidine bases in nucleic acids by environmental agents forms cyclobutane pyrimidine dimers. This happens DNA also may be damaged by reactive chemicals in- most frequently between adjacent thymidine residues troduced into the environment as products of industrial on the same DNA strand(Fig. 8-34). A second type of activity. Such products may not be injurious per se but pyrimidine dimer, called a 6-4 photoproduct, is also may be metabolized by cells into forms that are. Two formed during UV irradiation. lonizing radiation(x rays prominent classes of such agents(Fig. 8-35)are(1) and gamma rays) can cause ring opening and fragmen- deaminating agents, particularly nitrous acid (HNO2)or tation of bases as well as breaks in the covalent back- compounds that can be metabolized to nitro bone of nucleic acids nitrites, and(2)alkylating agents Virtually all forms of life are exposed to energy-ric Nitrous acid, formed from organic precursors such adiation capable of causing chemical changes in DNA. as nitrosamines and from nitrite and nitrate salts, is a Near-UV radiation(with wavelengths of 200 to 400 nm), potent accelerator of the deamination of bases. Bisulfite which makes up a significant portion of the solar spec- has similar effects. Both agents are used as preserva trum, is known to cause pyrimidine dimer formation and tives in processed foods to prevent the growth of toxic other chemical changes in the DNA of bacteria and of bacteria. They do not appear to increase cancer risks

cellular conditions. Depurination of ribonucleotides and RNA is much slower and generally is not considered physiologically significant. In the test tube, loss of purines can be accelerated by dilute acid. Incubation of DNA at pH 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid. Other reactions are promoted by radiation. UV light induces the condensation of two ethylene groups to form a cyclobutane ring. In the cell, the same reaction between adjacent pyrimidine bases in nucleic acids forms cyclobutane pyrimidine dimers. This happens most frequently between adjacent thymidine residues on the same DNA strand (Fig. 8–34). A second type of pyrimidine dimer, called a 6-4 photoproduct, is also formed during UV irradiation. Ionizing radiation (x rays and gamma rays) can cause ring opening and fragmen￾tation of bases as well as breaks in the covalent back￾bone of nucleic acids. Virtually all forms of life are exposed to energy-rich radiation capable of causing chemical changes in DNA. Near-UV radiation (with wavelengths of 200 to 400 nm), which makes up a significant portion of the solar spec￾trum, is known to cause pyrimidine dimer formation and other chemical changes in the DNA of bacteria and of human skin cells. We are subject to a constant field of ionizing radiation in the form of cosmic rays, which can penetrate deep into the earth, as well as radiation emit￾ted from radioactive elements, such as radium, pluto￾nium, uranium, radon, 14C, and 3 H. X rays used in med￾ical and dental examinations and in radiation therapy of cancer and other diseases are another form of ionizing radiation. It is estimated that UV and ionizing radiations are responsible for about 10% of all DNA damage caused by environmental agents. DNA also may be damaged by reactive chemicals in￾troduced into the environment as products of industrial activity. Such products may not be injurious per se but may be metabolized by cells into forms that are. Two prominent classes of such agents (Fig. 8–35) are (1) deaminating agents, particularly nitrous acid (HNO2) or compounds that can be metabolized to nitrous acid or nitrites, and (2) alkylating agents. Nitrous acid, formed from organic precursors such as nitrosamines and from nitrite and nitrate salts, is a potent accelerator of the deamination of bases. Bisulfite has similar effects. Both agents are used as preserva￾tives in processed foods to prevent the growth of toxic bacteria. They do not appear to increase cancer risks 294 Chapter 8 Nucleotides and Nucleic Acids 3 (a) Deamination 3 2 2 2 2 CH N HN Hypoxanthine Uracil N Cytosine O CH O N N Xanthine O N N O HN NH NH N N N NH 5-Methylcytosine Thymine O HN O O N N N N Adenine Guanine O N H N N O N N H N N O HN HN FIGURE 8–33 Some well-characterized nonenzymatic reactions of nucleotides. (a) Deamination reactions. Only the base is shown. (b) Depurination, in which a purine is lost by hydrolysis of the N-
- glycosyl bond. The deoxyribose remaining after depurination is readily converted from the
-furanose to the aldehyde form (see Fig. 8–3). Fur￾ther nonenzymatic reactions are illustrated in Figures 8–34 and 8–35

8.3 Nucleic Acid Chemistry 295 FIGURE 8-34 Formation of pyrimidine dimers induced by UV light. (a) One type of reaction (on the left] results in the formation C=0 of a cyclobutyl ring involving C-5 and C-6 of adjacent pyrimidir residues. An altemative reaction (on the right) results in a 6-4 photoproduct, with a linkage between C-6 of one pyrimidine and d jacent C-4 of its neighbor. (b) Formation of a cyclobutane pyrimic dimer introduces a bend or kink into the dna C=0 T CH C=0 CH yimin am 6-4 Photoproduct significantly when used in this way, perhaps because Possibly the most important source of mutagenic al- they are used in small amounts and make only a minor terations in DNA is oxidative damage. Excited-oxygen contribution to the overall levels of dna damage. (The species such as hydrogen peroxide, hydroxyl radicals, potential health risk from food spoilage if these preser- and superoxide radicals arise during irradiation or as a vatives were not used is much greater.) byproduct of aerobic metabolism. Of these species, the Alkylating agents can alter certain bases of DNA. hydroxyl radicals are responsible for most oxidative For example, the highly reactive chemical dimethylsul- DNA damage. Cells have an elaborate defense system fate(Fig. 8-35b)can methylate a guanine to yield o- to destroy reactive oxygen species, including enzymes methylguanine, which cannot base-pair with cytosine. such as catalase and superoxide dismutase that convert reactive oxygen species to harmless products. A frac tion of these oxidants inevitably escape cellular de fenses, however, and damage to DNA occurs through any of a large, complex group of reactions ranging from H oxidation of deoxyribose and base moieties to strand breaks. Accurate estimates for the extent of this dam- Guanine age are not yet available, but every day the dna of each tautomer human cell is subjected to thousands of damaging oxidative reactions CHo This is merely a sampling of the best-understood reactions that damage DNA. Many carcinogenic com HON HNNN pounds in food, water, or air exert their cancer-causing effects by modifying bases in DNA. Nevertheless, the in- o'Metbylruanine tegrity of dNa as a polymer is better maintained than that of either RNA or protein, because DNA is the only Many similar reactions are brought about by alkylating macromolecule that has the benefit of biochemical repair agents normally present in cells, such as S-adenosyl- systems. These repair processes(described in Chapter 25) greatly lessen the impact of damage to DNA

significantly when used in this way, perhaps because they are used in small amounts and make only a minor contribution to the overall levels of DNA damage. (The potential health risk from food spoilage if these preser￾vatives were not used is much greater.) Alkylating agents can alter certain bases of DNA. For example, the highly reactive chemical dimethylsul￾fate (Fig. 8–35b) can methylate a guanine to yield O6 - methylguanine, which cannot base-pair with cytosine. Many similar reactions are brought about by alkylating agents normally present in cells, such as S-adenosyl￾methionine. Possibly the most important source of mutagenic al￾terations in DNA is oxidative damage. Excited-oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism. Of these species, the hydroxyl radicals are responsible for most oxidative DNA damage. Cells have an elaborate defense system to destroy reactive oxygen species, including enzymes such as catalase and superoxide dismutase that convert reactive oxygen species to harmless products. A frac￾tion of these oxidants inevitably escape cellular de￾fenses, however, and damage to DNA occurs through any of a large, complex group of reactions ranging from oxidation of deoxyribose and base moieties to strand breaks. Accurate estimates for the extent of this dam￾age are not yet available, but every day the DNA of each human cell is subjected to thousands of damaging oxidative reactions. This is merely a sampling of the best-understood reactions that damage DNA. Many carcinogenic com￾pounds in food, water, or air exert their cancer-causing effects by modifying bases in DNA. Nevertheless, the in￾tegrity of DNA as a polymer is better maintained than that of either RNA or protein, because DNA is the only macromolecule that has the benefit of biochemical repair systems. These repair processes (described in Chapter 25) greatly lessen the impact of damage to DNA. 8.3 Nucleic Acid Chemistry 295 OH C N H OH C O C C H N O C N H CH3 C O C C H N UV light UV light Adjacent thymines Cyclobutane thymine dimer 6-4 Photoproduct O C N H CH3 C O C C H N O C N H CH3 C O C C H N O C N H CH3 C O C C H N O C N H CH3 C O C C H N 4 6 CH3 5 6 5 6 (a) N P P P FIGURE 8–34 Formation of pyrimidine dimers induced by UV light. (a) One type of reaction (on the left) results in the formation of a cyclobutyl ring involving C-5 and C-6 of adjacent pyrimidine residues. An alternative reaction (on the right) results in a 6-4 photoproduct, with a linkage between C-6 of one pyrimidine and C-4 of its neighbor. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA

296 Chapter 8 Nucleotides and Nucle eic Acids neNO, NaNO Sodum ninie Sodumnirte C-且NH2 o R N-N0 Dimathyh tosmic Dimethy lanate Nitrosamine (a) Nitrous acid CH,-CH-CI OH OH FIGURE 8-35 Chemical agents that cause CH,-CH-CI DNA damage.(a) Precursors of nitrous acid, s-Adenceylmdhimine Ninan mustard which promotes deamination reactions (b)Alkylating agents tb)Alkylating agents Some Bases of DNA Are Methylated quence. Until the late 1970s, determining the sequence of a nucleic acid containing even five or ten nucleotides Certain nucleotide bases in DNA molecules are enzy- was difficult and very laborious. The development of two matically methylated. Adenine and cytosine are meth new techniques in 1977, one by Alan Maxam and Walter lated more often than guanine and thymine Methyla- Gilbert and the other by Frederick Sanger, has made pos- tion is generally confined to certain sequences or sible the sequencing of ever larger DNA molecules with regions of a DNA molecule. In some cases the function an ease unimagined just a few decades ago. The tech tion remains unclear. All known DNA methylases use s. niques depend on an improved understanding of nu adenosylmethionine as a methyl group donor. E. coli trophoretic methods for separating DNA strands differing has two prominent methylation systems. One serves as in size by only one nucleotide. Electrophoresis of DNA is part of a defense mechanism that helps the cell to dis. similar to that of proteins(see Fig. 3-19) Polyacrylamide tinguish its DNA from foreign DNa by marking is often used as the gel matrix in work with short DNA own DNA with methyl groups and destroying(foreign) molecules(up to a few hundred nucleotides ); agarose is DNA without the methyl groups(this is known as a restriction-modification system; see Chapter 9). Th generally used for longer pieces of DNA. other system methylates adenosine residues within the In both Sanger and Maxam-Gilbert sequencing, the general principle is to reduce the DNa to four sets of la- sequence (5")GATC(3")to N-methyladenosine(Fig. beled fragments. The reaction producing each set is methylation)methylase, a component of a system that base-specific, so the lengths of the fragments correspond 8-5a). This is mediated by the Dam(DNA adenine to positions in the dNa sequence where a certain base repairs mismatched base pairs formed occasionally dur- occurs. For example, for an oligonucleotide with the se- ing DNA replication(see Fig. 25-20) quence pAATCGACT, labeled at the 5'end(the left end) In eukaryotic cells, about 5% of cytidine residues in a reaction that breaks the DNA after each C residue will DNA are methylated to 5-methylcytidine(Fig. 8-5a) generate two labeled fragments: a four-nucleotide and a Methylation is most common at CpG sequences. pro- seven-nucleotide fragment: a reaction that breaks the ducing methyl-CpG symmetrically on both strands of the DNA after each G will produce only one labeled, five- DNA. The extent of methylation of CpG sequences nucleotide fragment. Because the fragments are radio- varies by molecular region in large eukaryotic DNA mol actively labeled at their 5'ends, only the fragment to the ecules. Methylation suppresses the migration of seg. 5, side of the break is visualized. The fragment sizes cor- ments of DNA called transposons, described in Chapter respond to the relative s of c and g residues in 25. These methylations of cytosine also have structural the sequence. When the sets of fragments corresponding significance. The presence of 5-methylcytosine in an al- to each of the four bases are electrophoretically sepa- ternating CpG sequence markedly increases the ten- rated side by side, they produce a ladder of bands from which the sequence can be read directly(Fig. 8-36). We The Sequences of Long DNA Strands illustrate only the Sanger method, because it has proven Can Be Determined to be technically easier and is in more widespread use It requires the enzymatic synthesis of a DNA strand com In its capacity as a repository of information, a DNA mol- plementary to the strand under analysis, using a radio- ecules most important property is its nucleotide actively labeled"primer and dideoxynucleotides

Some Bases of DNA Are Methylated Certain nucleotide bases in DNA molecules are enzy￾matically methylated. Adenine and cytosine are methy￾lated more often than guanine and thymine. Methyla￾tion is generally confined to certain sequences or regions of a DNA molecule. In some cases the function of methylation is well understood; in others the func￾tion remains unclear. All known DNA methylases use S￾adenosylmethionine as a methyl group donor. E. coli has two prominent methylation systems. One serves as part of a defense mechanism that helps the cell to dis￾tinguish its DNA from foreign DNA by marking its own DNA with methyl groups and destroying (foreign) DNA without the methyl groups (this is known as a restriction-modification system; see Chapter 9). The other system methylates adenosine residues within the sequence (5
)GATC(3
) to N6 -methyladenosine (Fig. 8–5a). This is mediated by the Dam (DNA adenine methylation) methylase, a component of a system that repairs mismatched base pairs formed occasionally dur￾ing DNA replication (see Fig. 25–20). In eukaryotic cells, about 5% of cytidine residues in DNA are methylated to 5-methylcytidine (Fig. 8–5a). Methylation is most common at CpG sequences, pro￾ducing methyl-CpG symmetrically on both strands of the DNA. The extent of methylation of CpG sequences varies by molecular region in large eukaryotic DNA mol￾ecules. Methylation suppresses the migration of seg￾ments of DNA called transposons, described in Chapter 25. These methylations of cytosine also have structural significance. The presence of 5-methylcytosine in an al￾ternating CpG sequence markedly increases the ten￾dency for that segment of DNA to assume the Z form. The Sequences of Long DNA Strands Can Be Determined In its capacity as a repository of information, a DNA mol￾ecule’s most important property is its nucleotide se￾quence. Until the late 1970s, determining the sequence of a nucleic acid containing even five or ten nucleotides was difficult and very laborious. The development of two new techniques in 1977, one by Alan Maxam and Walter Gilbert and the other by Frederick Sanger, has made pos￾sible the sequencing of ever larger DNA molecules with an ease unimagined just a few decades ago. The tech￾niques depend on an improved understanding of nu￾cleotide chemistry and DNA metabolism, and on elec￾trophoretic methods for separating DNA strands differing in size by only one nucleotide. Electrophoresis of DNA is similar to that of proteins (see Fig. 3–19). Polyacrylamide is often used as the gel matrix in work with short DNA molecules (up to a few hundred nucleotides); agarose is generally used for longer pieces of DNA. In both Sanger and Maxam-Gilbert sequencing, the general principle is to reduce the DNA to four sets of la￾beled fragments. The reaction producing each set is base-specific, so the lengths of the fragments correspond to positions in the DNA sequence where a certain base occurs. For example, for an oligonucleotide with the se￾quence pAATCGACT, labeled at the 5
end (the left end), a reaction that breaks the DNA after each C residue will generate two labeled fragments: a four-nucleotide and a seven-nucleotide fragment; a reaction that breaks the DNA after each G will produce only one labeled, five￾nucleotide fragment. Because the fragments are radio￾actively labeled at their 5
ends, only the fragment to the 5
side of the break is visualized. The fragment sizes cor￾respond to the relative positions of C and G residues in the sequence. When the sets of fragments corresponding to each of the four bases are electrophoretically sepa￾rated side by side, they produce a ladder of bands from which the sequence can be read directly (Fig. 8–36). We illustrate only the Sanger method, because it has proven to be technically easier and is in more widespread use. It requires the enzymatic synthesis of a DNA strand com￾plementary to the strand under analysis, using a radio￾actively labeled “primer” and dideoxynucleotides. 296 Chapter 8 Nucleotides and Nucleic Acids FIGURE 8–35 Chemical agents that cause DNA damage. (a) Precursors of nitrous acid, which promotes deamination reactions. (b) Alkylating agents

8.3 Nucleic Acid Chemistry dATP dgTP 3′ACGG Template FIGURE 8-36 DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25).(a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide In cells, the 3'-hy. s with an incoming deoxynucleoside w phosphodiester bond. (b)The Sanger eoxynucleoside triphosphate(ddNTP) CTAAGCTCGA CT sis. (The Sanger method is also known Tamplate ddNTP is inserted in place of a dNTP. the analog is added because if lacks dcTP, ddTP, dA TP, dTTp ded for the next step to be sequenced is used as the tem. and, and a short primer, radioactively or fluorescently labeled, is an aAA节 +dCTP + ddGTP ddtTp GattegAgctdO -carreau nealed to it. By addition of small amounts of a single ddNTP, for CADdy example ddCTP, to an otherwise reaction system, the synthesized normally occurs. Given the excess of that the analog will be incorporated 11 AaroaAoTTAo IC at some point during synthesis. The mixture of labeled fragments, each end. gth, such that the different-sized frag- esis, reveal the location of c residues ately for each of the four ddNTPs, an tly from an autoradiogram of the gel migrate faster, the fragments near the cleotide positions closest to the primer Autendgeam cf alanced complementary quence obtained is that of the strand ing analyzed

8.3 Nucleic Acid Chemistry 297 (a) P P dATP dGTP 3
P P P OH A P P P P P P P P C G G A T C OH A OH G P C P C P G P T 5
Primer strand Template strand P P C T FIGURE 8–36 DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3
-hy￾droxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond. (b) The Sanger sequencing procedure uses dideoxynucleoside triphosphate (ddNTP) analogs to interrupt DNA synthesis. (The Sanger method is also known as the dideoxy method.) When a ddNTP is inserted in place of a dNTP, strand elongation is halted after the analog is added, because it lacks the 3
-hydroxyl group needed for the next step. (c) The DNA to be sequenced is used as the tem￾plate strand, and a short primer, radioactively or fluorescently labeled, is an￾nealed to it. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some lo￾cations where dC normally occurs. Given the excess of dCTP over ddCTP, the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high proba￾bility of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each end￾ing with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the different-sized frag￾ments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5
end), and the sequence is read (in the 5
n 3
direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed