6
The Chemical Structure,
Replication, and
Manipulation of DNA
Genome Size
• Complex organisms have large
genomes=genetic contents of a cell
• Genomic size increases with evolutionary
complexity
• Size of DNA is measured in kb=kilobase
pairs
• Size of large genomes is measured in
Mb=megabase pairs
DNA: Chemical Composition
• Gene = unique sequence of DNA bases
• Two types of nitrogen-containing bases
comprise the chemical structure of DNA:
- purines = adenine and guanine
- pyrimidines = thymine and cytosine
DNA: Chemical Composition
• Hydrogen bonds between purines and
pyrimidines form the double-strand structure
of DNA
• Nucleotides = building blocks of DNA =
phosphate + sugar + base
• Nucleoside = sugar +base
• Sugar = 5 carbon
deoxyribose
• Phosphodiester bonds
link sugar molecules
to phosphate groups
DNA: Chemical Structure
• DNA nucleotides = a chain of bases
• Orientation of sugar-phosphate linkages = 5’
to 3’ as the phosphate
attached to the 5’ carbon
of one sugar is linked to
the 3’ carbon of the next
sugar
• Purine and pyrimidine
bases are linked to the
1’ carbon of sugar
DNA: Chemical Structure
• DNA consists of two polynucleotide chains
which run 5’ to 3’ in opposite
directions = antiparallel
• DNA chains are held together by
hydrogen bonds between bases
• DNA bases pair by Chargaff’s
rules:
- Adenine (A) pairs with
Thymine (T)
- Guanine (G) pairs with
Cytosine (C)
DNA: Watson-Crick Model
3-D structure of the DNA molecule:
• DNA is a double helix consisting of two
polynucleotide chains held together by
hydrogen bonds between
the purines and pyrimidines
• Helix forms major and
minor groove
• Diameter of the helix =
20 Angstroms
• Each turn of the helix =
10 bases = 34 Angstroms
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
DNA Replication
• Semi-conservative = each original DNA
strand is a template for a new strand
complementary to the original
• Meselson and Stahl showed that newly
synthesized DNA consists of one original
strand (unlabeled) and one new strand
labeled during synthesis with a heavy
isotope of nitrogen
DNA Replication
• Autoradiogram of replicating circular
chromosome shows that DNA synthesis
is bi-directional from a single start site =
origin of replication (OR)
• replication forks = region where parental
strands are separating and new strands
are synthesized
Circular DNA Replication
• Movement of the replication fork is aided
by topoisomerases = enzymes which
unwind the DNA helix to permit strand
separation
• DNA topoisomerase I unwinds DNA by
cutting one strand, rotating it around the
second strand and then sealing the single
strand break (nick)
Linear DNA Replication
• Replication of linear DNA molecules
proceeds bidirectionally from multiple
origins of replication which form
replication loops
• Replication continues to expand the
replication loops until they fuse to form
two separate molecules of DNA
• The replicated DNAs contain the same
DNA sequence
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
DNA Synthesis
• Addition of nucleotides into growing
DNA chain by DNA
polymerase occurs by cleavage of two
phosphate groups and the attachment of the
nucleoside monophosphate to the 3’-OH of
adjacent deoxyribose sugar
• Complementary base pairing with template
specifies new strand order
DNA Synthesis
• DNA polymerase extends
a chain of nucleotides in
5’- to- 3’ direction only
• Template strand is
antiparallel
• DNA polymerase proofreading function =
3’-to-5’ exonuclease which removes
mismatched bases
DNA Synthesis
Each replication fork consists of:
• Leading strand: continuous synthesis
• Lagging strand: discontinuous synthesis
• DNA polymerase
synthesizes lagging
strand in short
segments = Okazaki
fragments
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
• RNA = primer to initiate DNA synthesis at
origins of replication
Primers: Role in Replication
• Primer = short RNA segment complementary
to DNA at origins of replication synthesized
by primase
• Primer provides free 3’-OH which can be
extended by DNA polymerase
• Okazaki fragments are
also initiated by primers
eventually replaced by
DNA; DNA ligase joins
ends
DNA Replication: Proteins
• Topoisomerases: nick and unwind DNA to
permit strand separation
• RNA primase: initiates strand synthesis by
forming RNA primer
• Helicase: unwinds DNA at replication fork
• Single-strand binding protein: stabilize
DNA at replication fork
DNA Replication: Proteins
• DNA polymerase complex : catalyzes the
incorporation of DNA nucleotides n 3’-to-5’
direction
• DNA ligase: joins Okazaki fragments on
lagging strand
Nucleic Acid Hybridization
• DNA denaturation = strand separation occurs
by heat to break hydrogen bonds between
DNA bases
• DNA renaturation =
hybridization =
complementary single
strands pair and
hydrogen bonds form
• Hybridization does not
occur unless DNA bases
are complementary
Restriction Enzymes
• Restriction enzymes make
site specific cleavages in
each DNA strand to generate
“nicked” single strands with
new 5’ and 3’ ends
• Many enzymes cut each
DNA strand at different base sites with
“staggered cleavages” creating short
unpaired “sticky” ends
Southern Blot Analysis
• DNA bands on a gel can often be visualized
by staining with dyes which bind DNA
(ethidium bromide)
• Southern blot analysis is used to detect very
small amounts of DNA or to identify a single
DNA band
• Southern blots use
labeled “probes” to
identify bands by
hybridization to
complementary
DNA bases
Southern Blot Analysis
Steps in Southern blot procedure:
• DNA is cut into pieces by restriction
enzymes
• DNA fragments are separated by gel
electrophoresis
• DNA is transferred from gel to
hybridization filter =blot procedure and
denatured to produce single-strand bands
of DNA
Southern Blot Analysis
• Filter is mixed with radiolabeled singlestranded DNA probe complementary to
the DNA sequence at high temperatures
which permit hybridization = hydrogen
bonds form between complementary base
pairs
• DNA bands hybridized to probe are
detected by X-ray film exposure
Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR) is used to
detect and amplify very small amounts of
DNA
• DNA sequence to be
amplified is targeted
using
• primer oligonucleotides
= short synthetic singlestranded DNA segments
complementary to the
DNA sequence flanking
the region to be amplified
Polymerase Chain Reaction
• Multiple short cycles of replication
occurring within the region flanked by
primers occurs by controlled temperature
shifts which result in repetitive rounds of:
- primer hybridization
- DNA replication of the targeted region by
primer extension
- strand separation
Polymerase Chain Reaction
• DNA polymerase used in PCR = Taq
polymerase isolated from bacterial
thermophiles which can withstand high
temperature used in procedure
• PCR accomplishes the rapid production of
large amounts of target DNA which can
then be identified and analyzed
DNA Sequence Analysis
• DNA sequence analysis determines the order
of bases in DNA
• Dideoxy method uses DNA bases containing
modified deoxyribose sugars = dideoxyribose
which contain H at
the 3’ position of the
ribose sugar rather
than OH
• Modified sugars
cause chain
termination
Dideoxy Method: DNA
Sequencing
• Each of four reactions contains a different
dideoxynucleotide =
A, T, G, or C in addition
to the four bases
• Synthesis occurs in each
reaction tube until a dideoxy
base is inserted which
results in chain termination
• Each tube contains a set
of DNA pieces ending with
the same base
Dideoxy Method: DNA
Sequencing
• Gel electrophoresis is used to separate
the reaction products from each tube =
DNAs end in A, G, T or C
• DNA sequence can be read in the 5’-to-3’
direction from the bottom of gel
• Each band on the gel is one base longer
than the previous band
• Bases are identified by gel position