Chapter 9 Nucleotides and Nucleic Acids

1. The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are polymers of nucleotide units1.1 DNA consists of four kinds of deoxyribonucleotide units linked together through covalent bonds

1.1.1 Each nucleotide unit is made of a nitrogenous base (the various part in the four different deoxyribonucleotides), a pentose sugar, and a phosphate group.

1.1.2 The nitrogenous base can be adenine (A), guanine (G), cytosine (C), or thymine (T) (uracil (U) in RNA).

1.1.3 The nitrogenous bases are derivatives of two parent compounds, pyrimidine and purine.

1.1.4 The carbon and nitrogen atoms in the pyrimidine and purine rings are numbered. (fig.)

1.1.5 The pentose in a deoxyribonucleotide is a deoxyribose, which lacks an oxygen atom at the 2’-position that is present in ribose, the parent compound. (the numbering of the sugar ring).

1.1.6 The deoxyribose is in its -furanose form (a closed five-member ring).

1.1.7 Only D-deoxyribose (the asymmetric carbon farthest to the carbonyl group has the same configuration as D-glyceraldehyde) is found in DNA.

1.1.8 Each pyrimidine is covalently linked (through a N-glycosidic bond) to the 1’ carbon of the deoxyribose at N-1 of the pyrimidine, and each purine is covalently linked to the 1’ carbon of the deoxyribose at N-9 of the purine.

1.1.9 The configuration of this N-glycosidic bond is , where the base lies on the same side of the furanose ring as the 5’ carbon.

1.1.10 The phosphate group is esterified to the -OH group on the 5’ carbon of the deoxyribose ring.

1.1.11 A nucleotide lacking the phosphate part is called a nucleoside.

1.1.12 The four nucleoside units in DNA are called deoxyadenosine, deoxyquanosine, deoxythymidine, and deoxycytidine.

1.1.2 The nitrogenous base can be adenine (A), guanine (G), cytosine (C), or thymine (T) (uracil (U) in RNA).

1.1.13 The four nucleotide units in DNA are called deoxyadensine 5’-monophosphate (dAMP, or deoxyadenylate), deoxyguanosine 5’-monophosphate (dGMP, or deoxyguanylate), deoxythymidine 5’-monophosphate (dTMP, or deoxythymidylate), and deoxycytidine 5’-monophosphate (dCMP, or deoxycytidylate).

1.2 RNA also consists of four different kinds of ribonucleotides.

1.2.1 Each ribonucleotide unit is also made of three parts: a nitrogenous base, a pentose, and a phosphate group.

1.2.2 The base part is adenine, guanine, cytosine or uracil.

1.2.3 Uracil exists only in RNA, and thymine only in DNA.

1.2.4 The pentose part is a ribose (without being deoxygenated at the 2’ position) in its -furanose form (as deoxyribose in deoxyribonucleotides).

1.2.5 The bases and the phosphate group are covalently linked to the ribose ring in the same ways as in deoxyribonucleotides.

1.2.6 The four nucleoside units in RNA are called adenosine, guanosine, cytidine, and uridine (without deoxy- suffix); and the nucleotide units are AMP, GMP, CMP, and UMP.

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1.3 The only known function of DNA is store genetic information.

1.3.1 The amino acid sequence of every protein and the nucleotide sequence of every RNA molecule in a cell are all specified by the nucleotide sequence of that cell’s DNA molecule.

1.3.2 A segment of DNA that contains the information required for the synthesis of a functional protein or RNA is referred as a gene.

1.3.3 DNA is large biomacromolecule. In bacteria, all the genetic information is stored in a single DNA molecule; in a eukaryotic cell each chromosome contains one single DNA molecule.

1.4 RNA can be divided into several classes of different functions.

1.4.1 Ribosomal RNAs (rRNA) are structural components of ribosomes (the protein synthesis machine in cells).

1.4.2 Messenger RNA (mRNA) are copies of DNA (synthesized by DNA transcription), that carry the information of one or a few genes to the ribosomes, where the corresponding protein(s) is(are) synthesized.

1.4.3 Transfer RNA (tRNA) are adapter molecules that faithfully translate the information in a mRNA molecule into the specific amino acid sequences in a polypeptide chain.

1.4.4 Some RNA molecules, named as Ribozymes, have catalytic activities functioning in the processing (cleavage) of precursor RNA molecules (Thomas Cech and Sidney Altman won the Nobel Prize in Chemistry in 1989 for discovering ribozymes).

1.5 Some bases are modified in both DNA and RNA molecules.

1.5.1 The most common modification found in DNA are methylation of some bases (catalyzed by specific DNA methylases or methyltransferase), including, e.g., N6-Methyladenine, 5-methylcytosine, N2-methylguanine)

1.5.2 The higher level of 5-methylcytosine in certain eukaryotic DNA sequences (often at CpG sequences) correspond to a lower level of gene activities.

1.5.3 In bacteria, certain bases on the genomic DNA are methylated to distinguish it from foreign DNA (as a result, the restriction enzymes produced in bacteria can cleave the invading foreign DNA).

1.5.4 Some minor bases are found in tRNA molecules, including, e.g., hypoxanthine, pseudouracil, 7-methylguanine, and 4-thioluracil.

1.6 Nucleotides have roles other than being monomeric units of nucleic acids.

1.6.1 Nucleoside triphosphates are used as source of chemical energy to drive a wide variety of biochemical reactions.

1.6.2 ATP is the “energy currency” in cells (UTP, GTP, and CTP are also used in specific reactions as energy sources)

1.6.3 Adenosine diphosphate (ADP) is part of many coenzymes, e.g., coenzyme A, nicotinamide adenine dinucleotide (NAD+), flavin adenine dinucleotide (FAD).

1.6.4 Adenosine 3’,5’-cyclic monophosphate (cAMP), guanosine 3’,5’-cyclic monophosphate (cGMP) function as secondary messengers in cell signal transductions.

2. Phosphodiester bonds link successive nucleotides in nucleic acids (in both DNA and RNA)

2.1 The 3’-hydroxyl group of one nucleotide is joined to the 5’-hydroxyl group of the next nucleotide by a phosphodiester bridge.

2.1.1 The covalent backbones of nucleic acids consist of alternating phosphate and pentose (-D-deoxyribose in DNA, -D-ribose in RNA) residues.

2.1.2 The characteristic bases can be regarded as side groups attaching to the backbone at regular intervals (similar to the R groups on a peptide chains).

2.1.3 Each DNA and RNA strands have a specific polarity with a distinct 5’ end (the end lacking a nucleotide at the 5’ position) and a 3’ end (the end lacking a nucleotide at the 3’ position). 5’-pCpGpT-3’-OH

2.1.4 The base sequence of a DNA or RNA molecule is always written with the 5’ end on the left and 3’ end on the right by convention.

2.1.5 The nucleotide sequences of short segment of nucleic acids can be represented in different ways. (fig.)

2.1.6 An oligonucleotide refers to nucleic acids shorter than about 50 nucleotides.

2.1.7 The backbones of both DNA and RNA are hydrophilic, having negative charges at physiological pH, that are generally neutralized by positively charged proteins, metal ions, and polyamines(?) in cells.

2.2 RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not.

2.2.1 The 2’-hydroxyl group, which is lacking in DNA, is directly involved (as nucleophile) in the process.

2.2.2 The 2’,3’-cyclic monophosphate derivatives formed in the process are rapidly hydrolyzed to yield a mixture of 2’- and 3’- nucleoside monophosphates.

3. The pyrimidines and purines common in DNA and RNA are highly conjugated (resonant) molecules.3.1 The resonance involving many atoms in the base ring gives most of the bonds in the ring partial double-bond character.

3.1.1 The pyrimidine rings are planar and the purine rings are nearly planar (with a slight pucker).

3.2 Free pyrimidine and purine bases may exist in two or more tautomeric forms depending upon the pH.

3.2.1 Lactam, lactim, and double lactim forms are present at various pH. (fig.) 3.2.2 At physiological pH, the lactam form is dominant.3.3 All of the bases absorb UV light as a result of resonance.

3.3.1 Nucleic acids are characterized by a strong absorption at 260 nm.

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4. DNA was found to be the molecule storing the genetic information.

4.1 Fred Griffith discovered that a nonvirulent R form of pneumococcus bacterium (with rough colonies) can be transformed into the virulent S form (of smooth colonies).

4.1.1 Injecting a mixture of live R and heat-killed S form was lethal to the mice, whereas neither live R nor heat-killed S form was lethal to the mice.

4.1.2 The blood of the dead mice contain live S pneummococci.

4.1.3 This change (R to S transformation) is permanent: The transformed pneumococci yielded virulent progeny of the S form.

4.1.4 Some cells in a growing culture of the R form were found to be transformed into the S form by the addition of cell-free extract of heat-killed S pneumococci.

4.1.5 The “transformation principle” was not elucidated.

4.2 DNA was found to carry the genetic information for virulence in the pneumococci transformation experiment of Griffith.

4.2.1 Addition of DNA extracted from the heat-killed S form pneumococci (with protein removed as completely as possible, how?) into live nonvirulent R form bacteria transformed the R form into a virulent S form permanently. 4.2.2 Treatment with proteolytic enzymes (trypsin, chymotrypsin) did not have any effect on the transformation activity.

4.2.3 Treatment with ribonuclease (known to digest RNA) had no effect on the transformation activity.

4.2.4 Treatment with deoxyribonuclease (known to digest DNA) destroyed the transformation activity.

4.2.5 Chromosomal proteins were assumed to carry the genetic information (with DNA playing a secondary role) until Avery, MacLeod, McCarty performed these experiments in 1944.

4.3 Further support for the genetic role of DNA came from the studies of T2 bacteriophage (a bacterial virus) that infects E.coli.

4.3.1 The T2 bacteriophage consists of a core of DNA surrounded by a protein coat.

4.3.2 Alfred Hershey and Martha Chase demonstrated that at infection only DNA (labeled with radioisotope 32P) entered E.coli cells, proteins (labeled with 35S) did not enter the host cells (1952).

4.3.3 DNA provided the genetic information for bacteriophage replication within the E.coli cells.

4.4 The DNA content was found to be the same for all somatic cells that have a diploid set of chromosomes.

4.4.1 Haploid cells were found to have half as much DNA. (evidence?).

5. DNA molecules are double helices.5.1 The ratios of adenine to thymine and of guanine to cytosine were found to be nearly 1.0 in DNA samples from all species studied (by Erwin Chargaff, 1950).

5.1.1 In all DNA molecules the number of adenine residues is always equal to that of thymine, and the number of guanine is always equal to cytosine (“the Chargaff Rules”).

5.1.2 The meaning of this equivalence was not evident until James Watson and Francis Crick proposed the DNA double helix model (which, however, was used as one of the key clues for the establishment of the three-dimensional structure of DNA).

5.2 DNA exists as a regular two-chain structure with H-bonds formed between opposing bases on the two chains.

5.2.1 Watson and Crick proposed a model (by precise model building) on the three-dimensional structure of DNA molecules based mainly on three main pieces of evidence.

A) the fact that the DNA molecule is composed of bases, deoxyriboses, and phosphate groups linked together as a polydeoxyribonucleotide.

B) X-ray diffraction pattern of DNA fibers, suggesting a helical structure with two distinctive regularities of 3.4 and 34 Angstroms along the axis of the molecule.

C) Chargaff’s discovery on the quantitative relationships between the bases (A=T, G=C).

5.2.2 The DNA molecule is a right-handed double helix containing two antiparallel strands.

5.2.3 The phosphate-deoxyribose backbones are on the outside of the helix (forming a “hydrophilic surface”), whereas the purine and pyrimidine bases are stacked inside (the base-stacking interactions make a major nonspecific contribution to the stability of the duplex).

5.2.4 The planes of the bases are perpendicular to the helix and the planes of the deoxyribose rings are nearly at right angles to those of the bases.

5.2.5 The two antiparallel chains are complementary to each other through hydrogen bonds between pairs of bases. Adenine is always paired with thymine (with two H-bonds), guanine with cytosine (with three H-bonds).

5.2.6 The specific base-pairing was proposed on the bases of the “Chargaff rules”, optimal hydrogen bonding and optimal spacing (the A-T, G-C paired structure would make insufficient room for two purines, and more than enough space for two pyrimidines).

5.2.7 The diameter of the proposed helix is about 20 Å, adjacent bases are separated by 3.4 Å and related by a rotation of about 36 with the helical structure repeats about every 10 residues on each chain at intervals of about 34 Å.

5.2.8 The DNA molecule contains two kinds of grooves, a major groove (of ~12 Å wide) and a minor groove (of ~6 Å wide), formed because the glycosidic bonds of a base pair are not diametrically opposite to each other. (fig.)

5.2.9 The major groove display more distinctive potential H-bonding features than the minor groove (also the larger size of the major groove makes it more accessible for interactions with proteins that recognize specific DNA sequences).

5.3 The double-helical model of DNA immediately suggested a mechanism for the replication of DNA.

5.3.1 Genetic information has to be replicated (duplicated).

5.3.2 The double helix model for DNA is, in effect, a pair of templates, each of which is complementary to the other.

5.3.3 It was proposed that at replication, the parent strands become separated (H-bonds are broken), and each forms the template for biosynthesis of a complementary daughter strand.

5.3.4 The two double-helical DNA molecules are exactly the same as the parent duplex (genetic information is thus replicated).

5.3.5 The DNA duplex model accounted for all the available data and was later proved correct (with minor modifications).

5.3.6 Watson, Crick, and Wilkins shared the Nobel Prize in medicine or physiology in 1962 for this brilliant accomplishment.

5.3.7 The discovery of the DNA double helix revolutionized biology: it led the way to an understanding of gene function in molecular terms (their work is recognized to mark the beginning of molecular biology).

5.4 DNA can occur in different structural forms.5.4.1 DNA is remarkably flexible molecule

with many rotatable bonds (thermal fluctuations producing bending, stretching, and unpairing).

5.4.2 The duplex structure proposed by Watson and Crick is referred as the B-form DNA, and is found to be the most stable structure for a random nucleotide sequence under physiological conditions. Thus it is the standard structure for DNA molecules.

5.4.3 At reduced humidity the DNA molecule will take the A-form: it is still a right-handed duplex made up of antiparallel strands held together by Watson-Crick base pairing.

5.4.4 The A-form helix is wider and shorter than the B-form helix mainly resulted from the C3’-endo conformation in the deoxyribose rings (which makes the phosphate group closer to the pentose ring and binds less water molecules and also becomes wider and shorter).

5.4.5 The Z-form DNA is a left-handed double helix in which backbone phosphates zigzag.

5.4.6 The Z-form DNA is adopted by short oligonucleotides that have sequences of alternating pyrimidines and purines (e.g., CGCGCG).

5.4.7 The zigzagging is a consequence of the fact that the repeating unit is a dinucleotide rather than a mononucleotide.

5.4.8 The biological roles of Z-DNA is uncertain (may play roles in gene expression and genetic recombination).

5.5 Electron microscopic observation revealed that many DNA molecules are circular and supercoiled.

5.5.1 Intact DNA molecules from bacteria, some viruses, mitochondria, and chloroplasts are circular.

5.5.2 The axis of the double helix can be twisted to form a superhelix (the circular DNA without any superhelical turns is known as a relaxed molecule).

5.5.3 Supercoiling makes the DNA molecule more compact thus important for its packaging in cells (also sediment more rapidly).

5.5.4 Interconversion of isomers having various degrees of supercoiling is catalyzed by topoisomerases (existing in prokaryotes and eukaryotes).

5.5.5 figures and other definitions (e.g., linking numbers).

6. Certain DNA sequences adopt unusual structures.

6.1 Some sequence cause bends in the DNA helix.

6.1.1 Bends are produced when four or more continuous adenine residues exist.

6.1.2 DNA bending may be important for protein binding (may also be protein-binding induced?).

6.2 Palindrome sequences have the potential to form hairpins or cruciforms (fig.).

6.2.1 Palidrome sequences have two twofold rotational symmetry.

6.2.2 Such sequences are self-complementary within each of the two strands, thus having the potential to form hairpin or cruciform structure.

6.2.3 DNA sequences specifically recognized by many restriction enzymes are palindromes. (proteins have similar twofold symmetry, e.g., by forming a dimer).

6.3 Triple-helical structures form when one DNA strand contains only long stretches of pyrimidines.

6.3.1 The triple-helical DNA is called the H-form DNA.

6.3.2 Two of the strands in the triple helix structure contain pyrimidines and the third purines.

6.3.3 Watson-Crick and non-Watson-Crick (Hoogsteen) H-bondings exist among the three strands.

6.3.4 Sequences that can form triple helix structures are found within regions of DNA involved in the regulation of expression of many genes in eukaryotes. (current progress?).

7. RNA molecules do not form simple, regular secondary structure but many form complex and unique three dimensional structures.7.1 Single strand RNA tends to take right-handed helical conformation.

7.1.1 Base stacking interactions is dominating in taking up this conformation. 7.2 Self-complementary sequences on a RNA molecule lead to specific structures.

7.2.1 The standard base-pairing rules are followed (i.e., A with U, G with C).

7.2.2 Base pairing between G and U is also common in RNA (represented by a dot).

7.2.3 Intrastrand base pairing makes structures including bulges, internal loops, and hairpins (helical).

7.2.4 The base paired double strand DNA segments take the structure similar to the A-form of DNA, while the B-form structure has not been observed.

7.2.5 The Z-form helices have been made in the laboratory under very high salt and high temperature conditions.

7.3 The three dimensional structure of tRNA has been determined by X-ray crystallography.

7.3.1 tRNA is “L”-like in shape with functional regions (anticodon bases and acceptor end) locating at the ends.

7.3.2 Phosphate and hydroxyl groups on the pentose rings participate in H-bonding.

7.3.3 The three dimensional structure of tRNA molecules is very much like that of proteins: unique and complex.

7.3.4 Recent results from RNAp and others (Jane Doudner, rules about metal binding, etc.).

8. Duplex DNA and RNA molecules can be denatured and renatured.

8.1 Duplex nucleic acids unwind to form two single strands at extreme pH and high temperature with changed physical properties.

8.1.1 Viscosity decreases sharply.8.1.2 UV absorption at 260 nm increases signif

icantly, an effect called hyperchromism or hyperchromic effect. (base stacking decreases absorption?!).

8.1.3 The unwinding (i.e., denaturation) of the double helix is called melting because it occurs abruptly at a certain temperature (indicating that DNA duplex is a highly cooperative structure, held together by many reinforcing bonds including mainly the base stacking and base pairing).

8.1.4 Each species of DNA has a characteristic melting temperature (Tm or tm) at which half of the duplex chain is separated (fig.).

8.1.5 Tm of a DNA molecule depends markedly on its base composition: DNA with higher content of GC base pairs has higher Tm because there are three H-bonds between each GC base pair, but only two H-bonds between the AT pair (Tm has an approximate linear relationship with (G+C)%).

8.1.6 DNA segments rich in AT base pairs are melted first (at lower temperatures).

8.2 When the temperature or pH is returned to the biological range, the two separated complementary strands will spontaneously rewind (renature) to form a duplex structure.

8.2.1 This renaturation process is called annealing.

8.2.2 Annealing can occur between complementary DNA, RNA, or DNA-RNA hybrids.

8.2.3 Two single strand nucleic acids (DNA or RNA) having partial complementary sequences will anneal to form hybrid duplexes. This hybridization principle is widely used in detecting existence of specific DNA or RNA species in cells.

8.2.4 In Southern blotting, genomic DNA is isolated, digested with restriction enzymes, separated on agarose gel and then blotted with specific single strand DNA probes (analogous to Western blotting).

8.2.5 In Northern blotting, whole cellular RNA is isolated and separated on agarose gel, and then blotted with DNA or RNA probes.

9. Mutations can be produced by several types of changes in the base sequence of DNA.

9.1 Mutations are alterations in DNA structure that lead to permanent changes in the genetic information encoded.

9.1.1 The accumulation of mutations is probably intimately linked to the processes of aging and cancer.9.2 Nucleotides and nucleic acids undergo slow nonenzymatic transformations.

9.2.1 Several bases undergo spontaneous loss of their exocyclic amine groups (deamination): e.g., cytosine can be deaminated into uracil, adenine to hypoxanthine, guanine to xanthine.

9.2.2 The existence of thymine, instead of uracil in DNA makes the long-term storage of genetic information possible (if DNA contains uracil normally, it would be unlikely that uracil formed from spontaneous deamination of cytosine can be recognized and repaired!).

9.2.3 The glycosidic bonds in deoxyribonucleotides can be spontaneously hydrolyzed to form apurinic or apyrimidinic acids (which occurs much faster for purines than for pyrimidines).

9.2.4 Specific repair systems exist in cells to correct these damaging changes.

9.3 Certain types of radiation generate damages in DNA molecules.

9.3.1 UV light induces neighboring pyrimidines (especially thymines) to form covalent dimers which will introduce a kink on the DNA chain, making the DNA neither be able to be replicated nor to be copied into mRNA.

9.3.2 Pyrimidine dimers are continuously repaired through enzyme actions.

9.3.3 Ionizing radiation, including X-rays and -rays, can cause ring opening, fragmentation of bases, and breaks in the covalent backbone of nucleic acids, thus very damaging. (free radical mechanism?).

9.4 DNA may be damaged by various reactive chemical agents.

9.4.1 Nitrous acid (HNO2) and bisulfite accelerate the deamination of bases like cytosine, adenine, and guanine.

9.4.2 Alkylating agents, like dimethylsulfate, add alkyl groups to bases (e.g., the enol tautomer of guanine), which change the base pairing patterns of the bases.

9.4.3 Excited-oxygen species (including H2O2, hydroxyl radicals, superoxide radicals), generated from irradiation or aerobic metabolism, oxidize many groups causing strand breaking in DNA molecules.

9.5 Some base analogs can be incorporated into DNA causing mutations.

9.5.1 5-bromouracil (thymine analog) and 2-aminopurine (adenine analog) when incorporated into DNA will be base paired to guanine and cytosine, respectively, thus causing mutations at replication. (?)

9.6 Many carcinogenic compounds present in the environment cause cancers by damaging DNA.

9.6.1 There is a comprehensive DNA repair system in cells that greatly lessen the impact of all kinds of damage to DNA.

9.6.2 Repair systems for damaged RNA and proteins molecules have not been found.

10. The nucleotide sequences of a DNA molecule is usually determined by Sanger’s dideoxy termination method.10.1 The invention of DNA sequencing methods (in the late 1970s) were a result of improved understanding of nucleotide chemistry, DNA metabolism (especially DNA replication), and high-resolution electrophoresis methods.

10.1.1 Obtaining the nucleotide sequence of a short oligonucleotide was extremely difficult and very laborious before the unimaged easy Maxam-Gilbert (chemical cleavage) and Sanger (dideoxy) methods were invented.

10.1.2 Both methods were based on the general principle of reducing the DNA to be sequenced to four sets of base-specific labeled segments.

10.1.3 The key to the establishment of the chemical cleavage method (i.e., the Maxam-Gilbert method) is being able to label the end of a single strand DNA with 32P (with polynucleotide kinase) and find appropriate chemical methods to cleave at specific bases.

10.1.4 The key for the Sanger method is to terminate the enzyme catalyzed replication process (where the DNA fragment to be sequenced is used as the template) by adding specific 2’,3’-dideoxyribonucleotides (ddNTPs).

10.1.5 Single stranded DNA fragments differing in size by one nucleotide can be separated on denaturing (with 8 M urea) polyacrylamide gel electrophoresis.

10.2 The Sanger method for DNA sequencing is usually used for its technical simplicity.

10.2.1 An oligonucleotide primer (easily obtained by automatic solid phase synthesis, a method very similar to that of solid phase synthesis of peptides) is needed for the template-directed DNA synthesis.

10.2.2 dNTPs are all added in each of the four set of reactions, where the synthesis of the complementary strand is catalyzed by DNA polymerase I.

10.2.3 One specific ddNTP of small amount is added in each set of reaction to terminate chain extension reaction at specific kind of bases (whenever a dideoxy analog is added, the chain extension is terminated).

10.2.4 The four sets of reaction products are separated on polyacrylamide gel lanes side by side.

10.2.5 The synthesized DNA strand is detected by radioisotope labeling (by incorporating 32P- or 35

S-labeled dNTPs in the extension reaction, or label the 5’ end of the primer with 32P by polynucleotide kinase).

10.2.6 The nucleotide sequence of the complementary strand (of the strand to be sequenced) can be read directly from a sequencing gel.

10.2.7 Walter Gilbert and Frederick Sanger won the Nobel Prize in Chemistry in 1980 for their inventions.

10.3 DNA sequencing is automated based on a variation of Sanger’s dideoxy termination method.

10.3.1 Each of the four set of reactions has a primer labeled with fluorescent tags of different colors.

10.3.2 DNA products of all four sets of reactions are mixed and run on one lane of gel.

10.3.3 The migration order (reflecting the nucleotide sequence of the synthesized complementary strand) of various bands (each representing DNA products of certain length) is recorded by laser beams.

10.4 The goals of the genome projects are to determine all the nucleotide sequences of the complete DNA molecules that encode all the genetic information of various species.

10.4.1 The Human Genome Project is to determine the complete nucleotide sequence (of about 3X109 base pairs) of the DNA molecules encoding all the genetic information for the making of a human being.

10.4.2 The sequence technology has been advanced considerably (at least 10 times as fast as before) during the pursuit of this project. (technical details are not forthcoming).