Chapter 28 Nucleosides, Nucleotides, and Nucleic Acids Dr. Wolf's CHM 424 4

28.1 Pyrimidines and Purines Dr. Wolf's CHM 424 4

Pyrimidines and Purines In order to understand the structure and properties of DNA and RNA, we need to look at their structural components. We begin with certain heterocyclic aromatic compounds called pyrimidines and purines. Dr. Wolf's CHM 424

Pyrimidines and Purines Pyrimidine and purine are the names of the parent compounds of two types of nitrogen-containing heterocyclic aromatic compounds. N H 1 2 3 4 5 7 8 9 6 Pyrimidine Purine Dr. Wolf's CHM 424

Pyrimidines and Purines Amino-substituted derivatives of pyrimidine and purine have the structures expected from their names. N H H2N H N NH2 4-Aminopyrimidine 6-Aminopurine Dr. Wolf's CHM 424

Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms. N H HO N H O enol keto Dr. Wolf's CHM 424

Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms. H N OH keto H N O enol Dr. Wolf's CHM 424

Important Pyrimidines Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA. HN N H O HN N H O CH3 NH2 HN O N H Uracil Thymine Cytosine Dr. Wolf's CHM 424

Adenine and guanine are the principal purines of both DNA and RNA. Important Purines Adenine and guanine are the principal purines of both DNA and RNA. N NH2 N H O HN N H N H2N Adenine Guanine Dr. Wolf's CHM 424

Caffeine and Theobromine Caffeine (coffee) and theobromine (coffee and tea) are naturally occurring purines. Caffeine N O H3C CH3 Theobromine O HN N CH3 Dr. Wolf's CHM 424

28.2 Nucleosides Dr. Wolf's CHM 424 4

Nucleosides The classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside of D-ribofuranose or 2-deoxy-D-ribofuranose. Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of almost any carbohydrate. The purine or pyrimidine part of a nucleoside is referred to as a purine or pyrimidine base. Dr. Wolf's CHM 424

Cytidine occurs in RNA; its 2-deoxy analog occurs in DNA Table 28.2 Pyrimidine nucleosides NH2 HO OH O HOCH2 N Cytidine Cytidine occurs in RNA; its 2-deoxy analog occurs in DNA Dr. Wolf's CHM 424

Thymidine occurs in DNA Table 28.2 Pyrimidine nucleosides O N NH HO H3C HOCH2 Thymidine Thymidine occurs in DNA Dr. Wolf's CHM 424

Pyrimidine nucleosides Table 28.2 Pyrimidine nucleosides HOCH2 O N NH OH HO Uridine Uridine occurs in RNA Dr. Wolf's CHM 424

Adenosine occurs in RNA; its 2-deoxy analog occurs in DNA Table 28.2 Purine nucleosides HOCH2 O OH HO N NH2 Adenosine Adenosine occurs in RNA; its 2-deoxy analog occurs in DNA Dr. Wolf's CHM 424

Guanosine occurs in RNA; its 2-deoxy analog occurs in DNA Table 28.2 Purine nucleosides HOCH2 N NH O HO NH2 OH Guanosine Guanosine occurs in RNA; its 2-deoxy analog occurs in DNA Dr. Wolf's CHM 424

Nucleotides are phosphoric acid esters of nucleosides. Dr. Wolf's CHM 424 4

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid. OCH2 P HO O OH N NH2 5' 1' 2' 3' 4' Dr. Wolf's CHM 424

Adenosine Diphosphate (ADP) OCH2 OH N NH2 Dr. Wolf's CHM 424

Adenosine Triphosphate (ATP) ATP is an important molecule in several biochemical processes including: energy storage (Sections 28.4-28.5) phosphorylation O P HO OCH2 OH N NH2 Dr. Wolf's CHM 424

ATP and Phosphorylation HOCH2 O OH HO ATP + This is the first step in the metabolism of glucose. hexokinase ADP + O OH HO (HO)2POCH2 Dr. Wolf's CHM 424

Cyclic adenosine monophosphate (cAMP) cAMP and cGMP Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. CH2 O OH N NH2 P HO Cyclic adenosine monophosphate (cAMP) Dr. Wolf's CHM 424

Cyclic guanosine monophosphate (cGMP) cAMP and cGMP Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. CH2 N NH O NH2 OH O P HO Cyclic guanosine monophosphate (cGMP) Dr. Wolf's CHM 424

28.4 Bioenergetics Dr. Wolf's CHM 424 4

Bioenergetics is the thermodynamics of biological processes. Emphasis is on free energy changes (DG) when DG is negative, reaction is spontaneous in the direction written when DG is 0, reaction is at equilibrium when DG is positive, reaction is not spontaneous in direction written Dr. Wolf's CHM 424

Standard Free Energy (DG°)  mA(aq) nB(aq) Sign and magnitude of DG depends on what the reactants and products are and their concentrations. In order to focus on reactants and products, define a standard state. The standard concentration is 1 M (for a reaction in homogeneous solution). DG in the standard state is called the standard free-energy change and given the symbol DG°. Dr. Wolf's CHM 424

Standard Free Energy (DG°)  mA(aq) nB(aq) Exergonic: An exergonic reaction is one for which the sign of DG° is negative. Endergonic: An exergonic reaction is one for which the sign of DG° is positive. Dr. Wolf's CHM 424

Standard Free Energy (DG°)  mA(aq) nB(aq) It is useful to define a special standard state for biological reactions. This special standard state is one for which the pH = 7. The free-energy change for a process under these conditions is symbolized as DG°'. Dr. Wolf's CHM 424

28.5 ATP and Bioenergetics Dr. Wolf's CHM 424 4

G°' for hydrolysis of ATP to ADP is –31 kJ/mol ATP + H2O ADP + HPO42– G°' for hydrolysis of ATP to ADP is –31 kJ/mol Relative to ADP + HPO42–, ATP is a "high-energy" compound. When coupled to some other process, the conversion of ATP to ADP can provide the free energy to transform an endergonic process to an exergonic one. Dr. Wolf's CHM 424

Glutamic Acid to Glutamine + NH4+ –OCCH2CH2CHCO– +NH3 O DG°' = +14 kJ Reaction is endergonic + H2O H2NCCH2CH2CHCO– +NH3 O Dr. Wolf's CHM 424

Glutamic Acid to Glutamine –OCCH2CH2CHCO– + NH4+ + ATP +NH3 Reaction becomes exergonic when coupled to the hydrolysis of ATP + HPO42– H2NCCH2CH2CHCO– +NH3 O DG°' = –17 kJ + ADP Dr. Wolf's CHM 424

Glutamic Acid to Glutamine –OCCH2CH2CHCO– + ATP +NH3 Mechanism involves phosphorylation of glutamic acid OCCH2CH2CHCO– +NH3 O + ADP P –O Dr. Wolf's CHM 424

Glutamic Acid to Glutamine H2NCCH2CH2CHCO– +NH3 O + HPO42– followed by reaction of phosphorylated glutamic acid with ammonia OCCH2CH2CHCO– +NH3 O + NH3 P –O Dr. Wolf's CHM 424

28.6 Phosphodiesters, Oligonucleotides, and Polynucleotides Dr. Wolf's CHM 424 4

Phosphodiesters A phosphodiester linkage between two nucleotides is analogous to a peptide bond between two amino acids. Two nucleotides joined by a phosphodiester linkage gives a dinucleotide. Three nucleotides joined by two phosphodiester linkages gives a trinucleotide, etc. (See next slide) A polynucleotide of about 50 or fewer nucleotides is called an oligonucleotide. Dr. Wolf's CHM 424

Fig. 28.1 The trinucleotide ATG free 5' end free 3' end A T G phosphodiester linkages between 3' of one nucleotide and 5' of the next Dr. Wolf's CHM 424

Nucleic acids are polynucleotides. Dr. Wolf's CHM 424 4

Nucleic acids first isolated in 1869 (Johann Miescher) Oswald Avery discovered (1945) that a substance which caused a change in the genetically transmitted characteristics of a bacterium was DNA. Scientists revised their opinion of the function of DNA and began to suspect it was the major functional component of genes. Dr. Wolf's CHM 424

Moreover: %A = %T, and %G = %C Composition of DNA Erwin Chargaff (Columbia Univ.) studied DNAs from various sources and analyzed the distribution of purines and pyrimidines in them. The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) varied among species. But the total purines (A and G) and the total pyrimidines (T and C) were always equal. Moreover: %A = %T, and %G = %C Dr. Wolf's CHM 424

Composition of Human DNA For example: Purine Pyrimidine Adenine (A) 30.3% Thymine (T) 30.3% Guanine (G) 19.5% Cytosine (C) 19.9% Total purines: 49.8% Total pyrimidines: 50.1% Dr. Wolf's CHM 424

Structure of DNA James D. Watson and Francis H. C. Crick proposed a structure for DNA in 1953. Watson and Crick's structure was based on: •Chargaff's observations •X-ray crystallographic data of Maurice Wilkins and Rosalind Franklin •Model building Dr. Wolf's CHM 424

28.8 Secondary Structure of DNA: The Double Helix Dr. Wolf's CHM 424 4

Base Pairing Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding. 2-deoxyribose A T Dr. Wolf's CHM 424

Base Pairing Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding. Dr. Wolf's CHM 424

Base Pairing Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding. 2-deoxyribose G C Dr. Wolf's CHM 424

Base Pairing Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding. Dr. Wolf's CHM 424

•Gives proper Chargaff ratios (A=T and G=C) The DNA Duplex Watson and Crick proposed a double-stranded structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other. •Gives proper Chargaff ratios (A=T and G=C) •Because each pair contains one purine and one pyrimidine, the A---T and G---C distances between strands are approximately equal. •Complementarity between strands suggests a mechanism for copying genetic information. Dr. Wolf's CHM 424

Fig. 28.4 Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases. Dr. Wolf's CHM 424

Fig. 28.5 Helical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside. Dr. Wolf's CHM 424

28.9 Tertiary Structure of DNA: Supercoils Dr. Wolf's CHM 424 4

Random coiling would reduce accessibility to critical regions. DNA is coiled A strand of DNA is too long (about 3 cm in length) to fit inside a cell unless it is coiled. Random coiling would reduce accessibility to critical regions. Efficient coiling of DNA is accomplished with the aid of proteins called histones. Dr. Wolf's CHM 424

DNA winds around histone proteins to form nucleosomes. Histones Histones are proteins rich in basic amino acids such as lysine and arginine. Histones are positively charged at biological pH. DNA is negatively charged. DNA winds around histone proteins to form nucleosomes. Dr. Wolf's CHM 424

Linker contains about 50 base pairs. Histones Each nucleosome contains one and three-quarters turns of coil = 146 base pairs. Linker contains about 50 base pairs. Dr. Wolf's CHM 424

Histones Histone proteins + Supercoiled DNA Nucleosome = Dr. Wolf's CHM 424

28.10 Replication of DNA Dr. Wolf's CHM 424 4

Fig. 28.8 DNA Replication The DNA to be copied is a double helix, shown here as flat for clarity. The two strands begin to unwind. (next slide) Dr. Wolf's CHM 424

Fig. 28.8 DNA Replication Each strand will become a template for construction of its complement. Dr. Wolf's CHM 424

Fig. 28.8 DNA Replication Two new strands form as nucleotides that are complementary to those of the original strands are joined by phosphodiester linkages. Polynucleotide chains grow in the 5'-3' direction—continuous in the leading strand, discontinuous in the lagging strand. Dr. Wolf's CHM 424

Fig. 28.8 DNA Replication Two duplex DNAs result, each of which is identical to the original DNA. Dr. Wolf's CHM 424

Elongation of the growing DNA chain The free 3'-OH group of the growing DNA chain reacts with the 5'-triphosphate of the appropriate nucleotide. Dr. Wolf's CHM 424

Poly- nucleotide chain Fig. 28.9 Chain elongation OH CH2 O P O– Adenine, Guanine, Cytosine, or Thymine •• OH CH2OPO O Adenine, Guanine, Cytosine, or Thymine O– • Poly- nucleotide chain Dr. Wolf's CHM 424

Poly- nucleotide chain Fig. 28.9 Chain elongation O P O– – OH CH2 O P O– Adenine, Guanine, Cytosine, or Thymine CH2OPO • Poly- nucleotide chain Dr. Wolf's CHM 424

28.11 Ribonucleic Acids Dr. Wolf's CHM 424 4

DNA and Protein Biosynthesis According to Crick, the "central dogma" of molecular biology is: "DNA makes RNA makes protein." Three kinds of RNA are involved. messenger RNA (mRNA) transfer RNA (tRNA) ribosomal RNA (rRNA) There are two main stages. transcription translation Dr. Wolf's CHM 424

This complementary RNA is messenger RNA (mRNA). Transcription In transcription, a strand of DNA acts as a template upon which a complementary RNA is biosynthesized. This complementary RNA is messenger RNA (mRNA). Mechanism of transcription resembles mechanism of DNA replication. Transcription begins at the 5' end of DNA and is catalyzed by the enzyme RNA polymerase. Dr. Wolf's CHM 424

Fig. 28.10 Transcription Only a section of about 10 base pairs in the DNA is unwound at a time. Nucleotides complementary to the DNA are added to form mRNA. Dr. Wolf's CHM 424

There are three nucleotides per codon. The Genetic Code The nucleotide sequence of mRNA codes for the different amino acids found in proteins. There are three nucleotides per codon. There are 64 possible combinations of A, U, G, and C. The genetic code is redundant. Some proteins are coded for by more than one codon. Dr. Wolf's CHM 424

First letter Second letter Third letter Table 28.3 (p 1175) U C A G UUU Phe UCU Ser UAU Tyr UGU Cys U UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UCG Trp G U C A G Table 28.3 (p 1175) Dr. Wolf's CHM 424

U C A G UUU Phe UCU Ser UAU Tyr UGU Cys U UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UCG Trp G CUU Leu CCU Pro CAU His CGU Arg U CUC Leu CCC Pro CAC His CGC Arg C CUA Leu CCA Pro CAA Gln CGA Arg A CUG Leu CCG Pro CAG Gln CCG Arg G AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys ACG Arg G GUU Val GCU Ala GAU Asp GGU Gly U GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GCG Gly G Dr. Wolf's CHM 424

U C A G U C UAA Stop UGA Stop A UAG Stop G A G AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys ACG Arg G UAA, UGA, and UAG are "stop" codons that signal the end of the polypeptide chain. AUG is the "start" codon. Biosynthesis of all proteins begins with methionine as the first amino acid. This methionine is eventually removed after protein synthesis is complete. Dr. Wolf's CHM 424

There are 20 different tRNAs, one for each amino acid. Transfer tRNA There are 20 different tRNAs, one for each amino acid. Each tRNA is single stranded with a CCA triplet at its 3' end. A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA. Dr. Wolf's CHM 424

Example—Phenylalanine transfer RNA One of the mRNA codons for phenylalanine is: UUC 5' 3' AAG 3' 5' The complementary sequence in tRNA is called the anticodon. Dr. Wolf's CHM 424

Fig. 28.11 Phenylalanine tRNA OCCHCH2C6H5 + NH3 O Anticodon 3' 3' 5' 5' Dr. Wolf's CHM 424

Most of the RNA in a cell is ribosomal RNA Ribosomes are the site of protein synthesis. They are where translation of the mRNA sequence to an amino acid sequence occurs. Ribosomes are about two-thirds RNA and one-third protein. It is believed that the ribosomal RNA acts as a catalyst—a ribozyme. Dr. Wolf's CHM 424

28.12 Protein Biosynthesis Dr. Wolf's CHM 424 4

Protein Biosynthesis During translation the protein is synthesized beginning at its N-terminus. mRNA is read in its 5'-3' direction begins at the start codon AUG ends at stop codon (UAA, UAG, or UGA) Dr. Wolf's CHM 424

Methionine at N-terminus is present as its N-formyl derivative. Fig. 28.12 Translation Methionine at N-terminus is present as its N-formyl derivative. Reaction that occurs is nucleophilic acyl substitution. Ester is converted to amide. Dr. Wolf's CHM 424

Fig. 28.12 Translation Dr. Wolf's CHM 424

Ester at 3' end of alanine tRNA is Met-Ala. Fig. 28.12 Translation Ester at 3' end of alanine tRNA is Met-Ala. Process continues along mRNA until stop codon is reached. Dr. Wolf's CHM 424

28.13 AIDS Dr. Wolf's CHM 424 4

Acquired immune deficiency syndrome AIDS Acquired immune deficiency syndrome More than 22 million people have died from AIDS since disease discovered in 1980s Now fourth leading cause of death worldwide and leading cause of death in Africa (World Health Organization) Dr. Wolf's CHM 424

Several strains of HIV designated HIV-1, HIV-2, etc. Virus responsible for AIDS in people is HIV (human immunodeficiency virus) Several strains of HIV designated HIV-1, HIV-2, etc. HIV is a retrovirus. Genetic material is RNA, not DNA. Dr. Wolf's CHM 424

HIV reproduces and eventually infects other T4 lympocytes. HIV inserts its own RNA and an enzyme (reverse transcriptase) in T4 lymphocyte cell of host. Reverse transcriptase catalyzes the formation of DNA complementary to the HIV RNA. HIV reproduces and eventually infects other T4 lympocytes. Ability of T4 cells to reproduce decreases, interfering with bodies ability to fight infection. Dr. Wolf's CHM 424

AIDS Drugs AZT and ddI are two drugs used against AIDS that delay onset of symptoms. N O N3 H3C HOCH2 NH AZT N O HOCH2 NH ddI H Dr. Wolf's CHM 424

Protease inhibitors are used in conjunction with other AIDS drugs. Several HIV proteins are present in the same polypeptide chain and must be separated from each other in order to act. Protease inhibitors prevent formation of HIV proteins by preventing hydrolysis of polypeptide that incorporates them. Dr. Wolf's CHM 424

28.14 DNA Sequencing Dr. Wolf's CHM 424 4

Restriction enzymes cleave the polynucleotide to smaller fragments. DNA Sequencing Restriction enzymes cleave the polynucleotide to smaller fragments. These smaller fragments (100-200 base pairs) are sequenced. The two strands are separated. Dr. Wolf's CHM 424

Single stranded DNA divided in four portions. DNA Sequencing Single stranded DNA divided in four portions. Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. POCH2 OH O P HO base H Dr. Wolf's CHM 424

DNA Sequencing OH HO base P O POCH2 H The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP. POCH2 OH O P HO base H Dr. Wolf's CHM 424

DNA Sequencing Each tube also contains a "primer," a short section of the complementary DNA strand, labeled with radioactive phosphorus (32P). DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template. DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain. Dr. Wolf's CHM 424

There are four lanes on the electrophoresis gel. DNA Sequencing The contents of each tube are separated by electrophoresis and analyzed by autoradiography. There are four lanes on the electrophoresis gel. Each DNA fragment will be one nucleotide longer than the previous one. Dr. Wolf's CHM 424

Figure 27.29 Sequence of fragment ddA ddT ddG ddC T TG TGA TGAC TGACA TGACAT TGACATA TGACATAC TGACATACG TGACATACGT Dr. Wolf's CHM 424

Figure 27.29 Sequence of fragment Sequence of original DNA ddA ddT ddG ddC T TG TGA TGAC TGACA TGACAT TGACATA TGACATAC TGACATACG TGACATACGT A AC ACT ACTG ACTGT ACTGTA ACTGTAT ACTGTATG ACTGTATGC ACTGTATGCA Dr. Wolf's CHM 424

28.15 The Human Genome Project Dr. Wolf's CHM 424 4

Human Genome Project In 1988 National Research Council (NRC) recommended that the U.S. undertake the mapping and sequencing of the human genome. International Human Genome Sequencing Consortium (led by U.S. NIH) and Celera Genomics undertook project. Orginally competitors, they agreed to coordinate efforts and published draft sequences in 2001. Dr. Wolf's CHM 424

28.16 DNA Profiling and the Polymerase Chain Reaction Dr. Wolf's CHM 424 4

DNA sequencing involves determining the nucleotide sequence in DNA. DNA Profiling DNA sequencing involves determining the nucleotide sequence in DNA. The nucleotide sequence in regions of DNA that code for proteins varies little from one individual to another, because the proteins are the same. Most of the nucleotides in DNA are in "noncoding" regions and vary significantly among individuals. Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual. Dr. Wolf's CHM 424

PCR amplifies DNA by repetitive cycles of the following steps. When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") portions of it. PCR amplifies DNA by repetitive cycles of the following steps. 1. Denaturation 2. Annealing ("priming") 3. Synthesis ("extension" or "elongation") Dr. Wolf's CHM 424

Figure 28.14 (PCR) (a) Consider double-stranded DNA containing a polynucleotide sequence (the target region) that you wish to amplify. Target region (b) Heating the DNA to about 95°C causes the strands to separate. This is the denaturation step. Dr. Wolf's CHM 424

Figure 28.14 (PCR) (c) Cooling the sample to ~60°C causes one primer oligonucleotide to bind to one strand and the other primer to the other strand. This is the annealing step. (b) Heating the DNA to about 95°C causes the strands to separate. This is the denaturation step. Dr. Wolf's CHM 424

Figure 28.14 (PCR) (c) Cooling the sample to ~60°C causes one primer oligonucleotide to bind to one strand and the other primer to the other strand. This is the annealing step. (d) In the presence of four DNA nucleotides and the enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesis step and is carried out at 72°C. Dr. Wolf's CHM 424

This completes one cycle of PCR. Figure 28.14 (PCR) This completes one cycle of PCR. (d) In the presence of four DNA nucleotides and the enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesis step and is carried out at 72°C. Dr. Wolf's CHM 424

This completes one cycle of PCR. Figure 28.14 (PCR) This completes one cycle of PCR. (e) The next cycle begins with the denaturation of the two DNA molecules shown. Both are then primed as before. Dr. Wolf's CHM 424

(f) Elongation of the primed fragments completes the second PCR cycle. Figure 28.14 (PCR) (f) Elongation of the primed fragments completes the second PCR cycle. (e) The next cycle begins with the denaturation of the two DNA molecules shown. Both are then primed as before. Dr. Wolf's CHM 424

(f) Elongation of the primed fragments completes the second PCR cycle. Figure 28.14 (PCR) (f) Elongation of the primed fragments completes the second PCR cycle. (g) Among the 8 DNAs formed in the second cycle are two having the structure shown. Dr. Wolf's CHM 424

Figure 28.14 (PCR) The two contain only the target region and and are the ones that increase disproportionately in subsequent cycles. (g) Among the 8 DNAs formed in the second cycle are two having the structure shown. Dr. Wolf's CHM 424

Cycle Total DNAs Contain only target 0 (start) 1 0 1 2 0 2 4 0 3 8 2 Table 28.4 Cycle Total DNAs Contain only target 0 (start) 1 0 1 2 0 2 4 0 3 8 2 4 16 8 5 32 22 10 1,024 1,004 20 1,048,566 1,048,526 30 1,073,741,824 1,073,741,764 Dr. Wolf's CHM 424

End of Chapter 28 Dr. Wolf's CHM 424 4