Chapter 9 Nucleotides and
Nucleic Acids
1,The nucleic acids,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),are polymers of
nucleotide units
1.1 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 b-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 b,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 b-
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.
Figure 10-1
Figure 10-2
Figure 10-3
Figure 10-4
Figure 10-5
Figure 10-6
Figure 10-7
Figure 10-8
Figure 10-10
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 (b-D-
deoxyribose in DNA,b-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.
Figure 10-9
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 ?,adjacent bases are separated by 3.4 ?
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 ?.
5.2.8 The DNA molecule contains two kinds
of grooves,a major groove (of ~12 ? wide) and a
minor groove (of ~6 ? 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 significantly,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 g-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
35S-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).
Nucleic Acids
1,The nucleic acids,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),are polymers of
nucleotide units
1.1 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 b-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 b,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 b-
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.
Figure 10-1
Figure 10-2
Figure 10-3
Figure 10-4
Figure 10-5
Figure 10-6
Figure 10-7
Figure 10-8
Figure 10-10
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 (b-D-
deoxyribose in DNA,b-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.
Figure 10-9
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 ?,adjacent bases are separated by 3.4 ?
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 ?.
5.2.8 The DNA molecule contains two kinds
of grooves,a major groove (of ~12 ? wide) and a
minor groove (of ~6 ? 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 significantly,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 g-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
35S-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).
