Chapter 6
DNA Structure, Replication, and
Manipulation
1
Genome Size
• The genetic complement of a cell or virus constitutes
its genome.
• In eukaryotes, this term is commonly used to refer to
one complete haploid set of chromosomes, such as
that found in a sperm or egg.
• The C-value is the DNA content of the haploid
genome
• The units of length of nucleic acids in which genome
sizes are expressed :
• kilobase (kb) 103 base pairs
• megabase (Mb) 106 base pairs
2
Genome Size
• Viral genomes are typically in the range 100–1000 kb:
– Bacteriophage MS2, one of the smallest viruses, has only four
genes in a single stranded RNA molecule of about 4000
nucleotides (4kb)
• Bacterial genomes are larger, typically in the range
1–10 Mb:
– The chromosome of Escherichia coli is a circular DNA molecule
of 4600 kb.
3
Genome Size
• Eukaryotic genomes are typically in the range 100–
1000 Mb:
–
The genome of a fruit fly, Drosophila melanogaster is 180 Mb
• Among eukaryotes, genome size often differs
tremendously, even among closely related species
4
The C-value Paradox
• Genome size among species of protozoa differ by
5800-fold, among arthropods by 250-fold, fish 350fold, algae 5000-fold, and angiosperms 1000-fold.
• The C-value paradox: Among eukaryotes, there is
no consistent relationship between the C-value and
the metabolic, developmental, or behavioral
complexity of the organism
• The reason for the discrepancy is that in higher
organisms, much of the DNA has functions other
than coding for the amino acid sequence of proteins
5
DNA: Chemical Composition
• DNA is a linear polymer of four deoxyribonucleotides
• Nucleotides composed of 2'- deoxyribose (a fivecarbon sugar), phosphoric acid, and the four nitrogencontaining bases denoted A, T, G, and C
Figure 6.3: A typical nucleotide showing the three major components
6
DNA: Chemical Composition
• Two of the bases, A and G, have a double-ring
structure; these are called purines
• The other two bases,T and C, have a single-ring
structure; these are called pyrimidines
Figure 6.2: Chemical structures of adenine, thymine, guanine, and cytosine
7
DNA Structure
• The nucleotides are joined to form a polynucleotide
chain, in which the phosphate attached to the 5'
carbon of one sugar is linked to the hydroxyl group
attached to the 3' carbon of the next sugar in line
• The chemical bonds by which the sugar
components of adjacent nucleotides are linked
through the phosphate groups are called
phosphodiester bonds
8
Figure 6.4: Three nucleotides at the 5’-end of a single polynucleotide strand
9
DNA Structure
• The duplex molecule of DNA consists of two
polynucleotide chains twisted around one another to
form a right-handed helix in which the bases form
hydrogen bonds.
• Adenine pairs with thymine; guanine with cytosine
• A hydrogen bond is a weak bond
• The stacking of the base pairs on top of one another
also contribute to holding the strands together
• The paired bases are planar, parallel to one another, and
perpendicular to the long axis of the double helix.
10
DNA Structure
• The backbone of each strand
consists of deoxyribose sugars
alternating with phosphate
groups that link 5' carbon of one
sugar to the 3' carbon of the next
sugar in line
• The two polynucleotide strands
of the double helix run in
opposite directions
• The paired strands are said to be
antiparallel
Figure 6.7: DNA molecule showing the antiparallel
orientation of the complementary strands
11
DNA: Watson-Crick Model
3-D structure of the DNA molecule:
• Double helix forms major and minor grooves
• Diameter of the helix = 20 Angstroms
• Each turn of the helix = 10 bases = 34 Angstroms
12
Figure 6.5A: Illustration of DNA helix
Figure 6.5B: Computer
model of DNA helix
Part B Courtesy of Antony M. Dean, University of Minnesota
13
DNA Replication
Watson-Crick model of DNA
replication:
• Hydrogen bonds between DNA
bases break to allow strand separation
• Each DNA strand is a template for the
synthesis of a new strand
• Template (parental) strand determines
the sequence of bases in the new
strand (daughter): complementary
base pairing rules
Figure 6.8: Watson–Crick
model of DNA replication
14
DNA Replication
• In 1958 M. Meselson and F. Stahl showed that DNA
replication is semiconservative:
– The parental strands remain intact and serves as a template
for a new strand
Figure 6.9: Predictions of semiconservative DNA replication
15
Circular DNA Replication
• Autoradiogram of the intact replicating circular
chromosome of E. coli shows that
– DNA synthesis is bidirectional
– Replication starts from a single site called origin of replication
(OR)
• The region in which parental strands are separating
and new strands are being synthesized is called a
replication fork
16
Figure 6.12: The distinction between unidirectional and bidirectional DNA
replication
17
Rolling Circle Replication
• Some circular DNA molecules of a number of
bacterial and eukaryotic viruses, replicate by a
different mode called rolling-circle replication.
• One DNA strand is cut by a nuclease to produce a
3'-OH extended by DNA polymerase.
• The newly replicated strand is displaced from the
template strand as DNA synthesis continues.
• Displaced strand is template for complementary
DNA strand.
18
Figure 6.13: Rolling-circle replication
19
Replication of Linear DNA
• The linear DNA duplex in a eukaryotic chromosome
also replicates bidirectionally
• Replication is initiated at many sites along the DNA
• Multiple initiation is a means of reducing the total
replication time
20
Figure 6.14: Replicating DNA of D. melanogaster
21
Replication of Linear DNA
• In eukaryotic cell, origins of replication are about
40,000 bp apart, which allows each chromosome
to be replicated in 15 to 30 minutes.
• Because chromosomes do not replicate
simultaneously, complete replication of all
chromosomes in eukaryotes usually takes from 5
to 10 hours.
22
DNA Synthesis
• One strand of the newly made DNA, leading
strand, is synthesized continuously.
• The other, lagging strand is made in small
precursor fragments = Okazaki fragments
• The size of Okazaki fragments is 1000–2000 base
pairs in prokaryotic cells and 100–200 base pairs
in eukaryotic cells.
23
Figure 6.22: Short fragments in the replication fork
24
DNA vs. RNA
• DNA sugar = deoxyribose
RNA sugar = ribose
• RNA contains the pyrimidine uracil (U) in place of
thymine (T)
• DNA is double-stranded
• RNA is single-strand
• Short RNA fragment serves as a primer to initiate
DNA synthesis at origins of replication
25
DNA Replication: Proteins
• Gyrase = topoisomerase II introduces a doublestranded break ahead of the replication fork and
swivels the cleaved ends to relieve the stress of
helix unwinding
• Helicase unwinds DNA at replication fork to
separate the parental strands
• Single-strand binding protein (SSB) stabilizes
single strands of DNA at replication fork
26
DNA Replication: Proteins
• Multienzyme complex called primosome initiates
strand synthesis by forming RNA primer
• The enzyme DNA polymerase forms the
phosphodiester bond between adjacent
nucleotides in a new DNA acid chain in 5' to 3'
direction
• DNA polymerase has a proofreading function that
corrects errors in replication
27
DNA Replication: Proteins
• The final stitching together of the lagging strand
must require:
• Removal of the RNA primer
• Replacement with a DNA sequence
• Joining adjacent DNA fragments
• Primer removal and replacement in E. coli is
accomplished by a special DNA polymerase (Pol I)
that removes one ribonucleotide at a time
28
DNA Replication: Proteins
• In eukaryotes, the primer RNA is removed as an
intact unit by a protein called RPA (replication
protein A)
• DNA ligase catalyzes the formation of the final
bond connecting the two precursor
29
Figure 6.15: Role of proteins in DNA replication
30
Nucleic Acid Hybridization
• DNA denaturation: Two DNA strands can be
separated by heat without breaking
phosphodiester bonds
• DNA renaturation = hybridization: Two single
strands that are complementary or nearly
complementary in sequence can come together to
form a different double helix
• Single strands of DNA can also hybridize
complementary sequences of RNA
31
Figure 6.24: Nucleic acid hybridization
32
Restriction Enzymes
• Restriction enzymes cleave duplex DNA at
particular nucleotide sequences
• The nucleotide sequence recognized for cleavage
by a restriction enzyme is called the restriction site
of the enzyme
• In virtually all cases, the restriction site of a
restriction enzyme reads the same on both strands
• A DNA sequence with this type of symmetry is
called a palindrome
33
Restriction Enzymes
• Many restriction enzymes all cleave their
restriction site asymmetrically—at a different site
on the two DNA strands
• They create sticky ends = each end of the cleaved
site has a single-stranded overhang that is
complementary in base sequence to the other end
• Some restriction enzymes cleave symmetrically—
at the same site in both strands
• They yield DNA fragments that have blunt ends
34
Restriction Enzymes
• Because of the sequence specificity, a particular
restriction enzyme produces a unique set of
restriction fragments for a particular DNA molecule.
• Another enzyme will produce a different set of
restriction fragments from the same DNA molecule.
• A map showing the unique sites of cutting of a
particular DNA molecule by restriction enzyme is
called a restriction map
35
Figure 6.26: A. A. EcoRI enzyme; B. Electrophoresis gel of BamHI and
EcoRI; C. BamHI enzyme
36
Southern Blot Analysis
• DNA fragments on a gel can often be visualized by
staining with ethidium bromide, a dye that binds DNA
• Particular DNA fragments can be isolated by cutting
out the small region of the gel that contains the
fragment and removing the DNA from the gel.
• Specific DNA fragments are identified by
hybridization with a probe = a radioactive fragment of
DNA or RNA
• Southern blot analysis is used to detect very small
amounts of DNA or to identify a particular DNA band
by DNA-DNA or DNA-RNA hybridization
37
Southern Blot Analysis
Steps in Southern blot procedure:
 DNA is digested by restriction enzymes
 DNA fragments are separated by gel
electrophoresis
 DNA is transferred from gel to hybridization
filter = blot procedure
 DNA denatured to produce single-stranded DNA
38
Southern Blot Analysis
• Filter is mixed with radiolabeled single-stranded
DNA or RNA probe at high temperatures that permit
hybridization = formation of hydrogen bonds
between complementary base pairs
• DNA bands hybridized to a probe are detected by xray film exposure
39
Figure 6.27: Southern blot
40
Polymerase Chain Reaction
• Polymerase chain reaction (PCR) makes possible
the amplification of a particular DNA fragment
• Oligonucleotide primers that are complementary to
the ends of the target sequence are used in
repeated rounds of denaturation, annealing, and
DNA replication
• The number of copies of the target sequence
doubles in each round of replication, eventually
overwhelming any other sequences that may be
present
41
Polymerase Chain Reaction
• Special DNA polymerase is used in PCR = Taq
polymerase isolated from bacterial thermophiles that
can withstand high temperature used in procedure
• PCR accomplishes the rapid production of large
amounts of target DNA that can then be identified
and analyzed
42
Figure 6.28: Polymerase chain reaction (PCR) for amplification
43
DNA Sequence Analysis
• DNA sequence analysis
determines the order of
bases in DNA
• The dideoxy sequencing
method employs DNA
synthesis in the presence
of small amounts of
fluorescently labeled
nucleotides that contain
the sugar dideoxyribose
instead of deoxyribose
Figure 6.29: Structures of normal deoxyribose
and the dideoxyribose sugar used in DNA sequencing.
44
DNA Sequencing:
Dideoxy Method
• Modified sugars cause chain
termination because it lacks the
3'-OH group, which is essential
for attachment of the next
nucleotide in a growing DNA
strand
• The products of DNA synthesis
are then separated by
electrophoresis. In principle, the
sequence can be read directly
from the gel.
Figure 6.30: Dideoxy
method of DNA sequencing
45
DNA Sequencing: Dideoxy Method
• Each band on the gel is one base longer than the
previous band
• Each didyoxynucleotide is labeled by different
fluorescent dye
• G, black; A, green; T, red; C, purple
• As each band comes off the bottom of the gel,
the fluorescent dye that it contains is excited by
laser light, and the color of the fluorescence is
read automatically by a photocell and recorded
in a computer
46
Figure 6.31: Fluorescence pattern obtained from a DNA sequencing gel
47
DNA Sequencing
• Massively parallel sequencing yields billions of
base pairs per day.
48
Figure 6.32: A method for massively parallel DNA sequencing that captures
and amplifies DNA template strands on tiny beads
49