Chapter 28

Chapter 28 - Chapter 28 Nucleosides, Nucleotides, and...

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Unformatted text preview: Chapter 28 Nucleosides, Nucleotides, and Nucleic Acids Dr. Wolf's CHM 424 28- 1 28.1 Pyrimidines and Purines Dr. Wolf's CHM 424 28- 2 Pyrimidines and Purines In order to understand the structure and In properties of DNA and RNA, we need to look at their structural components. their We begin with certain heterocyclic aromatic We compounds called pyrimidines and purines. compounds Dr. Wolf's CHM 424 28- 3 Pyrimidines and Purines Pyrimidine and purine are the names of the Pyrimidine parent compounds of two types of nitrogenparent containing heterocyclic aromatic compounds. 6 N1 5 4 7 N 2 3 Pyrimidine Dr. Wolf's CHM 424 N 5 9 N H 4 8 6 N1 N 2 3 Purine 28- 4 Pyrimidines and Purines Amino-substituted derivatives of pyrimidine and Amino-substituted purine have the structures expected from their names. names. H NH2 H N N H H2N N H 4-Aminopyrimidine Dr. Wolf's CHM 424 N N N H H 6-Aminopurine 28- 5 Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines But exist in keto, rather than enol, forms. exist H H HO H H N N enol Dr. Wolf's CHM 424 H O N N H H keto 28- 6 Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines But exist in keto, rather than enol, forms. exist OH N H N O N N H NH N H N H N H enol Dr. Wolf's CHM 424 keto 28- 7 H Important Pyrimidines Important Pyrimidines that occur in DNA are cytosine and Pyrimidines thymine. Cytosine and uracil are the pyrimidines in RNA. pyrimidines O O NH2 CH3 HN HN HN O N H Uracil Dr. Wolf's CHM 424 O N H Thymine O N H Cytosine 28- 8 Important Purines Adenine and guanine are the principal purines Adenine of both DNA and RNA. of NH2 O N N N N H Adenine Dr. Wolf's CHM 424 N HN H2N N N H Guanine 28- 9 Caffeine and Theobromine Caffeine (coffee) and theobromine (coffee and Caffeine tea) are naturally occurring purines. tea) O H3C O N N N N CH3 Caffeine Caffeine Dr. Wolf's CHM 424 O CH3 N HN O CH3 N N CH3 Theobromine Theobromine 28- 10 28.2 Nucleosides Dr. Wolf's CHM 424 28- 11 Nucleosides The classical structural definition is that a The nucleoside is a pyrimidine or purine N-glycoside of D-ribofuranose or 2-deoxy-D-ribofuranose. Informal use has extended this definition to Informal apply to purine or pyrimidine N-glycosides of almost any carbohydrate. almost The purine or pyrimidine part of a nucleoside is The referred to as a purine or pyrimidine base. purine Dr. Wolf's CHM 424 28- 12 Table 28.2 Pyrimidine nucleosides NH2 N HOCH2 O Cytidine Cytidine HO N O OH Cytidine occurs in RNA; Cytidine its 2-deoxy analog occurs in DNA its Dr. Wolf's CHM 424 28- 13 Table 28.2 Pyrimidine nucleosides O H3C HOCH2 Thymidine Thymidine NH N O O HO Thymidine occurs in DNA Dr. Wolf's CHM 424 28- 14 Table 28.2 Pyrimidine nucleosides O NH Uridine Uridine HOCH2 HO N O O OH Uridine occurs in RNA Dr. Wolf's CHM 424 28- 15 Table 28.2 Purine nucleosides NH2 N Adenosine Adenosine HOCH2 O HO N N N OH Adenosine occurs in RNA; Adenosine its 2-deoxy analog occurs in DNA its Dr. Wolf's CHM 424 28- 16 Table 28.2 Purine nucleosides O N Guanosine Guanosine HOCH2 O HOCH HO N NH N NH2 OH Guanosine occurs in RNA; Guanosine its 2-deoxy analog occurs in DNA its Dr. Wolf's CHM 424 28- 17 28.3 Nucleotides Nucleotides are phosphoric acid esters of Nucleotides nucleosides. nucleosides. Dr. Wolf's CHM 424 28- 18 Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also Adenosine called 5'-adenylic acid. called NH2 N O HO 5' OCH P OCH2 O 4' HO 3' HO Dr. Wolf's CHM 424 N N N 1' 2' OH 28- 19 Adenosine Diphosphate (ADP) NH2 N O HO O OCH P O P OCH2 O HO N HO HO Dr. Wolf's CHM 424 N N OH 28- 20 Adenosine Triphosphate (ATP) ATP is an important molecule in several ATP biochemical processes including: biochemical energy storage (Sections 28.4-28.5) phosphorylation O HO P HO O O OCH O P O P OCH2 O HO N N N N HO HO Dr. Wolf's CHM 424 NH2 OH 28- 21 ATP and Phosphorylation ATP + HOCH2 HO HO O HO This is the first step in the This metabolism of glucose. metabolism hexokinase OH O ADP + Dr. Wolf's CHM 424 (HO)2POCH2 O HO HO HO OH 28- 22 cAMP and cGMP Cyclic AMP and cyclic GMP are Cyclic "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. and NH2 N CH2 O CH O O HO P O N N N OH Cyclic adenosine monophosphate (cAMP) Dr. Wolf's CHM 424 28- 23 cAMP and cGMP Cyclic AMP and cyclic GMP are Cyclic "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. and O O HO P O N CH2 O CH O N NH N NH2 OH Cyclic guanosine monophosphate (cGMP) Dr. Wolf's CHM 424 28- 24 28.4 Bioenergetics Dr. Wolf's CHM 424 28- 25 Bioenergetics Bioenergetics is the thermodynamics of Bioenergetics biological processes. biological Emphasis is on free energy changes (∆ G) when ∆ G is negative, reaction is when spontaneous in the direction written spontaneous when ∆ G is 0, reaction is at equilibrium when when ∆ G is positive, reaction is not when spontaneous in direction written spontaneous Dr. Wolf's CHM 424 28- 26 Standard Free Energy (∆ G°) G°) mA(aq) nB(aq) Sign and magnitude of ∆ G depends on what the reactants and products are and their concentrations. concentrations. In order to focus on reactants and products, In define a standard state. standard The standard concentration is 1 M (for a The reaction in homogeneous solution). reaction ∆ G in the standard state is called the standard free-energy change and given the symbol ∆ G°. Dr. Wolf's CHM 424 28- 27 Standard Free Energy (∆ G°) G°) mA(aq) nB(aq) Exergonic: An exergonic reaction is one for which the sign of ∆ G° is negative. Endergonic: An exergonic reaction is one for Endergonic: which the sign of ∆ G° is positive. Dr. Wolf's CHM 424 28- 28 Standard Free Energy (∆ G°) G°) mA(aq) nB(aq) It is useful to define a special standard state for biological reactions. for This special standard state is one for which This the pH = 7. the The free-energy change for a process under The these conditions is symbolized as ∆ G°'. °'. Dr. Wolf's CHM 424 28- 29 28.5 ATP and Bioenergetics Dr. Wolf's CHM 424 28- 30 Hydrolysis of ATP ATP + H2O ADP + HPO42– ∆ G°' for hydrolysis of ATP to ADP is –31 kJ/mol Relative to ADP + HPO42–, ATP is a "highenergy" compound. When coupled to some other process, the When conversion of ATP to ADP can provide the free energy to transform an endergonic process to an exergonic one. an Dr. Wolf's CHM 424 28- 31 Glutamic Acid to Glutamine O O – OCCH2CH2CHCO– + NH4+ + NH NH3 ∆ G°' = +14 kJ O Reaction is endergonic O H2NCCH2CH2CHCO– + H2 O + NH NH3 Dr. Wolf's CHM 424 28- 32 Glutamic Acid to Glutamine O O – OCCH2CH2CHCO– + NH NH3 ∆ G°' = –17 kJ O + NH4+ + ATP Reaction becomes exergonic when coupled to the hydrolysis of ATP O H2NCCH2CH2CHCO– + HPO42– + ADP + NH NH3 Dr. Wolf's CHM 424 28- 33 Glutamic Acid to Glutamine O O + ATP – OCCH2CH2CHCO– + NH NH3 O – O O P O Mechanism involves phosphorylation of glutamic phosphorylation acid acid O OCCH2CH2CHCO– – O O Dr. Wolf's CHM 424 + ADP + NH NH3 28- 34 Glutamic Acid to Glutamine O O H2NCCH2CH2CHCO– + NH NH3 O – O O P O + HPO42– followed by reaction of followed phosphorylated glutamic acid with ammonia with O OCCH2CH2CHCO– – O O Dr. Wolf's CHM 424 + NH3 + NH NH3 28- 35 28.6 Phosphodiesters, Phosphodiesters, Oligonucleotides, and Polynucleotides Polynucleotides Dr. Wolf's CHM 424 28- 36 Phosphodiesters A phosphodiester linkage between two phosphodiester nucleotides is analogous to a peptide bond between two amino acids. between Two nucleotides joined by a phosphodiester Two linkage gives a dinucleotide. linkage Three nucleotides joined by two Three phosphodiester linkages gives a trinucleotide, etc. (See next slide) etc. A polynucleotide of about 50 or fewer polynucleotide nucleotides is called an oligonucleotide. oligonucleotide. Dr. Wolf's CHM 424 28- 37 NH2 free 5' end N 5' HOCH2 O phosphodiester phosphodiester linkages between 3' of one nucleotide and 5' of the next next A T free 3' end N N O H3C O O P OCH2 O HO G Dr. Wolf's CHM 424 N Fig. 28.1 The The trinucleotide ATG ATG NH N O O O O P OCH2 O HO N N NH N 3' HO 28- 38 NH2 28.7 Nucleic Acids Nucleic acids are polynucleotides. Dr. Wolf's CHM 424 28- 39 Nucleic Acids Nucleic acids first isolated in 1869 (Johann Nucleic Miescher) Miescher) Oswald Avery discovered (1945) that a Oswald substance which caused a change in the genetically transmitted characteristics of a bacterium was DNA. bacterium Scientists revised their opinion of the function of Scientists DNA and began to suspect it was the major functional component of genes. functional Dr. Wolf's CHM 424 28- 40 Composition of DNA Erwin Chargaff (Columbia Univ.) studied DNAs Erwin from various sources and analyzed the distribution of purines and pyrimidines in them. distribution The distribution of the bases adenine (A), The guanine (G), thymine (T), and cytosine (C) varied among species. varied But the total purines (A and G) and the total But pyrimidines (T and C) were always equal. pyrimidines Moreover: %A = %T, and %G = %C Dr. Wolf's CHM 424 28- 41 Composition of Human DNA For example: Purine Pyrimidine Adenine (A) 30.3% Guanine (G) 19.5% Total purines: 49.8% Thymine (T) 30.3% Cytosine (C) 19.9% Total pyrimidines: 50.1% Dr. Wolf's CHM 424 28- 42 Structure of DNA James D. Watson and Francis H. C. Crick James proposed a structure for DNA in 1953. proposed Watson and Crick's structure was based on: •Chargaff's observations •X-ray crystallographic data of Maurice •X-ray Wilkins and Rosalind Franklin Wilkins •Model building Dr. Wolf's CHM 424 28- 43 28.8 Secondary Structure of DNA: The Double Helix Dr. Wolf's CHM 424 28- 44 Base Pairing Watson and Crick proposed that A and T were Watson present in equal amounts in DNA because of complementary hydrogen bonding. complementary H N N N N H O H CH3 N 2-deoxyribose N N O A Dr. Wolf's CHM 424 T 2-deoxyribose 28- 45 Base Pairing Watson and Crick proposed that A and T were Watson present in equal amounts in DNA because of complementary hydrogen bonding. complementary Dr. Wolf's CHM 424 28- 46 Base Pairing Likewise, the amounts of G and C in DNA were Likewise, equal because of complementary hydrogen bonding. bonding. H O N HN N 2-deoxyribose NH N N NH H G Dr. Wolf's CHM 424 N O 2-deoxyribose C 28- 47 Base Pairing Likewise, the amounts of G and C in DNA were Likewise, equal because of complementary hydrogen bonding. bonding. Dr. Wolf's CHM 424 28- 48 The DNA Duplex Watson and Crick proposed a double-stranded Watson structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other. bonded •Gives proper Chargaff ratios (A=T and G=C) •Because each pair contains one purine and Because one pyrimidine, the A---T and G---C distances between strands are approximately equal. •Complementarity between strands suggests a Complementarity mechanism for copying genetic information. mechanism Dr. Wolf's CHM 424 28- 49 O 3' Fig. 28.4 O O Two antiparallel Two strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases. bases. C 5' G O O O PO 3' O T 5' A O O O PO Ğ O T A 5' O O PO O O O G O C 5' O O Dr. Wolf's CHM 424 Ğ OP O 3' O 3' 5' O Ğ O 5' Ğ 5' OP O 3' O 3' O O OP O 3' O Ğ O 5' 3' 28- 50 Ğ Fig. 28.5 Helical structure of Helical DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside. outside. Dr. Wolf's CHM 424 28- 51 28.9 Tertiary Structure of DNA: Supercoils Dr. Wolf's CHM 424 28- 52 DNA is coiled A strand of DNA is too long (about 3 cm in strand length) to fit inside a cell unless it is coiled. length) Random coiling would reduce accessibility to Random critical regions. critical Efficient coiling of DNA is accomplished with the Efficient aid of proteins called histones. histones. Dr. Wolf's CHM 424 28- 53 Histones Histones are proteins rich in basic amino acids Histones such as lysine and arginine. such Histones are positively charged at biological pH. DNA is negatively charged. DNA winds around histone proteins to form DNA nucleosomes. nucleosomes. Dr. Wolf's CHM 424 28- 54 Histones Histones Each nucleosome contains one and three-quarters turns of coil = 146 base pairs. turns Linker contains about 50 base pairs. Dr. Wolf's CHM 424 28- 55 Histones Histones Nucleosome = Nucleosome Dr. Wolf's CHM 424 Histone proteins + Supercoiled DNA 28- 56 28.10 Replication of DNA Dr. Wolf's CHM 424 28- 57 Fig. 28.8 DNA Replication The DNA to be copied is a double helix, shown here as flat for clarity. 5' 3' T A A T G C G C C G A T T A C G C G T A G C A T A T T A C G 5' 3' The two strands begin to unwind. (next slide) Dr. Wolf's CHM 424 28- 58 Fig. 28.8 DNA Replication Each strand will become a template for construction of its complement. 3' 5' T A A T G C G C 3' C G A T T A C G C G T A G C A T A T T A C G 5' Dr. Wolf's CHM 424 28- 59 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. 3' 5' T A G G C A T C T C A' G C' 3' A 3' T C C G T A G G Polynucleotide chains grow in the 5'-3' direction—continuous in the leading strand, discontinuous in the lagging strand. Dr. Wolf's CHM 424 A A T' A T' T A' C G' 5' leading strand 5' C A' T lagging strand A' T T' C' AG 5' 3' 28- 60 Fig. 28.8 DNA Replication Two duplex DNAs result, each of which is identical to the original DNA. 5' 3' T A' A T' G C' G C' CA G' T' T A' C G' C G' T A' G C' A T' A T' T A' C G' 5' 3' + 5' 3' T' A A' T G' G' C' CCG 3' Dr. Wolf's CHM 424 A' T T' C' AG C' G T' G' AC A' T A' T T' A C' G 5' 28- 61 Elongation of the growing DNA chain The free 3'-OH group of the growing DNA chain The reacts with the 5'-triphosphate of the appropriate nucleotide. nucleotide. Dr. Wolf's CHM 424 28- 62 Fig. 28.9 Chain elongation OH Adenine, Guanine, Cytosine, or Thymine Adenine, Guanine, Cytosine, or Thymine Dr. Wolf's CHM 424 O O CH2 O P CH O O OP OP O– •• •• • OH • O O CH2OPO CH O– O– O– O– PolyPolynucleotide nucleotide chain chain 28- 63 Fig. 28.9 Chain elongation O Adenine, Guanine, Cytosine, or Thymine O CH2 CH Adenine, Guanine, Cytosine, or Thymine Dr. Wolf's CHM 424 O O O –O P OH OP O– O– O– P O– •O• •• O O CH2OPO CH O– PolyPolynucleotide nucleotide chain chain 28- 64 28.11 Ribonucleic Acids Dr. Wolf's CHM 424 28- 65 DNA and Protein Biosynthesis According to Crick, the "central dogma" of According molecular biology is: molecular "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 28- 66 Transcription In transcription, a strand of DNA acts as a In template upon which a complementary RNA is biosynthesized. This complementary RNA is messenger RNA messenger (mRNA). (mRNA). Mechanism of transcription resembles Mechanism mechanism of DNA replication. mechanism Transcription begins at the 5' end of DNA and is catalyzed by the enzyme RNA polymerase. RNA Dr. Wolf's CHM 424 28- 67 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 28- 68 The Genetic Code The nucleotide sequence of mRNA codes for The the different amino acids found in proteins. the There are three nucleotides per codon. There are 64 possible combinations of A, U, G, There and C. and The genetic code is redundant. Some proteins The are coded for by more than one codon. are Dr. Wolf's CHM 424 28- 69 U U UUU UUC UUA UUG Phe Phe Leu Leu UCU UCC UCA UCG C Ser Ser Ser Ser Ser UAU UAU UAC UAA UAG A Tyr Tyr Stop Stop G UGU UGC UGA UCG Cys Cys Stop Trp C First letter A Second letter Third letter G Dr. Wolf's CHM 424 Table 28.3 (p 1175) Table 28.3 (p 1175) 28- 70 U C A G U C A G U C A G U C A G U U C A UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val C UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Ser Ser Ser Ser Pro Pro Pro Pro Pro Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala A UAU UAU UAC UAA UAG CAU CAU CAC CAA CAG AAU AAU AAC AAA AAG GAU GAU GAC GAA GAG Tyr Tyr Stop Stop His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu G UGU UGC UGA UCG CGU CGC CGA CCG AGU AGC AGA ACG GGU GGC GGA GCG Cys Cys Stop Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly G Dr. Wolf's CHM 424 28- 71 U C A G U C A G U C A G U C A G U U C UAA, UGA, and UAG are "stop" codons that signal the end of the polypeptide chain. A UAA Stop UAG Stop C A AUU AUC AUA AUG Ile Ile Ile Met ACU ACC ACA ACG Thr Thr Thr Thr Thr AAU AAU AAC AAA AAG Asn Asn Lys Lys G U C UGA Stop A G U C A G AGU Ser U AGC Ser C AGA Arg A ACG Arg G U C A of all G AUG is the "start" codon. Biosynthesis proteins begins with methionine as the first amino G acid. This methionine is eventually removed after protein synthesis is complete. Dr. Wolf's CHM 424 28- 72 Transfer tRNA There are 20 different tRNAs, one for each There amino acid. amino Each tRNA is single stranded with a CCA triplet Each at its 3' end. at A particular amino acid is attached to the tRNA particular by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA. tRNA. Dr. Wolf's CHM 424 28- 73 Transfer RNA Example—Phenylalanine transfer RNA One of the mRNA codons for phenylalanine is: 5' UUC 3' The complementary sequence in tRNA is called the anticodon. the anticodon 3' Dr. Wolf's CHM 424 AAG 5' 28- 74 Fig. 28.11 Phenylalanine tRNA O OCCHCH2C6H5 3' NH3 + 3' 5' 5' Anticodon Dr. Wolf's CHM 424 28- 75 Ribosomal RNA Most of the RNA in a cell is ribosomal RNA Ribosomes are the site of protein synthesis. Ribosomes They are where translation of the mRNA translation sequence to an amino acid sequence occurs. sequence Ribosomes are about two-thirds RNA and onethird protein. It is believed that the ribosomal RNA acts as a It catalyst—a ribozyme. ribozyme. Dr. Wolf's CHM 424 28- 76 28.12 Protein Biosynthesis Dr. Wolf's CHM 424 28- 77 Protein Biosynthesis During translation the protein is synthesized During beginning at its N-terminus. beginning 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 28- 78 Fig. 28.12 Translation Methionine at N-terminus is present as its N-formyl derivative. Reaction that occurs is Reaction nucleophilic acyl substitution. Ester is converted to amide. converted Dr. Wolf's CHM 424 28- 79 Fig. 28.12 Translation Dr. Wolf's CHM 424 28- 80 Fig. 28.12 Translation Ester at 3' end of Ester alanine tRNA is Met-Ala. alanine Process continues along Process mRNA until stop codon is reached. is Dr. Wolf's CHM 424 28- 81 28.13 AIDS Dr. Wolf's CHM 424 28- 82 AIDS Acquired immune deficiency syndrome More than 22 million people have died from More AIDS since disease discovered in 1980s AIDS Now fourth leading cause of death worldwide Now and leading cause of death in Africa (World Health Organization) Health Dr. Wolf's CHM 424 28- 83 HIV Virus responsible for AIDS in people is HIV Virus (human immunodeficiency virus) (human Several strains of HIV designated HIV-1, HIV-2, Several etc. etc. HIV is a retrovirus. Genetic material is RNA, not HIV retrovirus Genetic DNA. DNA. Dr. Wolf's CHM 424 28- 84 HIV HIV inserts its own RNA and an enzyme HIV (reverse transcriptase) iin T4 lymphocyte cell of n host. host. Reverse transcriptase catalyzes the formation of Reverse DNA complementary to the HIV RNA. DNA HIV reproduces and eventually infects other T4 HIV lympocytes. lympocytes. Ability of T4 cells to reproduce decreases, Ability interfering with bodies ability to fight infection. interfering Dr. Wolf's CHM 424 28- 85 AIDS Drugs AZT and ddI are two drugs used against AIDS AZT that delay onset of symptoms. that O O H3C HOCH2 O O N N3 AZT Dr. Wolf's CHM 424 O NH N NH HOCH2 NN O O H H H H ddI 28- 86 AIDS Drugs Protease inhibitors are used in conjunction with Protease other AIDS drugs. other Several HIV proteins are present in the same Several polypeptide chain and must be separated from each other in order to act. each Protease inhibitors prevent formation of HIV Protease proteins by preventing hydrolysis of polypeptide that incorporates them. that Dr. Wolf's CHM 424 28- 87 28.14 DNA Sequencing Dr. Wolf's CHM 424 28- 88 DNA Sequencing Restriction enzymes cleave the polynucleotide Restriction to smaller fragments. to These smaller fragments (100-200 base pairs) These are sequenced. are The two strands are separated. Dr. Wolf's CHM 424 28- 89 DNA Sequencing Single stranded DNA divided in four portions. Each tube contains adenosine, thymidine, Each guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. of OH OH HO P O O P O OH O POCH2 POCH O HO Dr. Wolf's CHM 424 base O H 28- 90 DNA Sequencing The first tube also contains the 2,'3'-dideoxy analog of The adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP. the OH OH HO P O O P O OH O POCH2 POCH O H Dr. Wolf's CHM 424 base O H 28- 91 DNA Sequencing Each tube also contains a "primer," a short Each section of the complementary DNA strand, labeled with radioactive phosphorus (32P). labeled DNA synthesis takes place, producing a DNA complementary strand of the DNA strand used as a template. as DNA synthesis stops when a dideoxynucleotide DNA is incorporated into the growing chain. is Dr. Wolf's CHM 424 28- 92 DNA Sequencing The contents of each tube are separated by The electrophoresis and analyzed by autoradiography. autoradiography. There are four lanes on the electrophoresis gel. Each DNA fragment will be one nucleotide Each longer than the previous one. longer Dr. Wolf's CHM 424 28- 93 Figure 27.29 ddA ddT ddG ddC Sequence of Sequence fragment fragment T TG TGA TGAC T GACA T GACAT T GACAT A T GACAT AC TGACATACG TGACATACGT Dr. Wolf's CHM 424 28- 94 Figure 27.29 ACTG T GACA ACTGT T GACAT ACTGTA T GACAT A ACTGTAT T GACAT AC ACTGTATG TGACATACG ACTGTATGC TGACATACGT Dr. Wolf's CHM 424 ACT TGAC ddC AC TGA ddG A TG ddT Sequence of Sequence original DNA original T ddA Sequence of Sequence fragment fragment ACTGTATGCA 28- 95 28.15 The Human Genome Project Dr. Wolf's CHM 424 28- 96 Human Genome Project In 1988 National Research Council (NRC) In recommended that the U.S. undertake the mapping and sequencing of the human genome. mapping International Human Genome Sequencing International Consortium (led by U.S. NIH) and Celera Genomics undertook project. Orginally competitors, they agreed to coordinate efforts and published draft sequences in 2001. and Dr. Wolf's CHM 424 28- 97 28.16 DNA Profiling and the Polymerase Chain Reaction Dr. Wolf's CHM 424 28- 98 DNA Profiling DNA sequencing involves determining the DNA nucleotide sequence in DNA. nucleotide The nucleotide sequence in regions of DNA that The code for proteins varies little from one individual to another, because the proteins are the same. to Most of the nucleotides in DNA are in Most "noncoding" regions and vary significantly among individuals. among Enzymatic cleavage of DNA give a mixture of Enzymatic polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual. Dr. Wolf's CHM 424 28- 99 PCR When a sample of DNA is too small to be When sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies reaction ("amplify") portions of it. ("amplify") PCR amplifies DNA by repetitive cycles of the PCR following steps. following 1. Denaturation 2. Annealing ("priming") 3. Synthesis ("extension" or "elongation") Dr. Wolf's CHM 424 28- 100 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 strands step. step. Dr. Wolf's CHM 424 28- 101 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 strands step. step. Dr. Wolf's CHM 424 28- 102 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 the extended in its 3' direction. This is the synthesis extended step and is carried out at 72°C. Dr. Wolf's CHM 424 28- 103 Figure 28.14 (PCR) This completes one cycle of PCR. This (d) In the presence of four DNA nucleotides and the enzyme DNA polymerase, the primer is the extended in its 3' direction. This is the synthesis extended step and is carried out at 72°C. Dr. Wolf's CHM 424 28- 104 Figure 28.14 (PCR) This completes one cycle of PCR. This (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 28- 105 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 28- 106 Figure 28.14 (PCR) Figure (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. cycle Dr. Wolf's CHM 424 28- 107 Figure 28.14 (PCR) Figure The two contain only the target region and and are the ones that increase disproportionately and in subsequent cycles. in (g) Among the 8 DNAs formed in the second cycle are two having the structure shown. cycle Dr. Wolf's CHM 424 28- 108 Table 28.4 Cycle Total DNAs 0 (start) 1 1 2 2 4 3 8 4 16 5 32 10 1,024 20 1,048,566 30 1,073,741,824 Dr. Wolf's CHM 424 Contain only target 0 0 0 2 8 22 1,004 1,048,526 1,073,741,764 28- 109 End of Chapter 28 Dr. Wolf's CHM 424 28- 110 ...
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