1. Molecular Biology
Honors Biology
Edgar

17. DNA Replication

24. Exonuclease

27. Fig. 16-UN5

28. Fig. 16-13
Topoisomerase
Helicase
PrimaseSingle-strand binding
proteins
RNA
primer
5′
5′
5′ 3′
3′
3′

30. Fig. 16-16b6
Template
strand
5′
5′3′
3′
RNA primer 3′
5′
5′
3′
1
1
3′
3′
5′
5′
Okazaki
fragment
12
3′
3′
5′
5′
12
3′
3′
5′
5′
1
2
5′
5′
3′
3′
Overall direction of replication

32. Fig. 16-16a
Overview
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions
of replication
1
2

33. Helicase

34. Topoisomerase and Helicase

39. Fig. 20-3-1
Restriction site
DNA
Sticky end
Restriction enzyme
cuts sugar-phosphate
backbones.
5′
3′
3′
5′
1

40. Fig. 20-3-2
Restriction site
DNA
Sticky end
Restriction enzyme
cuts sugar-phosphate
backbones.
5′
3′
3′
5′
1
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
2
One possible combination

41. Fig. 20-3-3
Restriction site
DNA
Sticky end
Restriction enzyme
cuts sugar-phosphate
backbones.
5′
3′
3′
5′
1
One possible combination
Recombinant DNA molecule
DNA ligase
seals strands.
3
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
2

42. Fig. 20-9a
Mixture of
DNA mol-
ecules of
different
sizes
Power
source
Longer
molecules
Shorter
molecules
Gel
AnodeCathode
TECHNIQUE
1
2
Power
source
– +
+–

43. Fig. 20-9b
RESULTS

45. Fig. 20-10
Normal
allele
Sickle-cell
allele
Large
fragment
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
201 bp
175 bp
376 bp
(a) DdeI restriction sites in normal and
sickle-cell alleles of β-globin gene
Normal β-globin allele
Sickle-cell mutant β-globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI

47. Restriction Enzyme Lab
• HINTS:
• pMAP is 5615bp
• There are
– 2 PstI sites.
– 1 HpaI site.
– 1 SspI site
• Lambda DNA/PstI:
• You should not be
able to see beyond
the 805bp band.
• Fine the 11,490bp
and the 805bp as
reference.

48. Transcription and Translation

71. To studying theTo studying the Wolbachia within?Wolbachia within?
Credit: Mark Taylor

72. 16S rRNA (ribosomal RNA)16S rRNA (ribosomal RNA)
 Small ribosomal subunit involved in mRNA translation processSmall ribosomal subunit involved in mRNA translation process
 Ancient molecule, conserved function, universally distributedAncient molecule, conserved function, universally distributed
 Helps identify unknown bacterium to genus or species levelsHelps identify unknown bacterium to genus or species levels
 Present in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cellsPresent in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cells
 Plays a catalytic and structural role in the ribosomePlays a catalytic and structural role in the ribosome

74. DNA Barcoding

76. Cytochrome Oxidase (COI)

77. Bacteria 16S rRNA and EukaryoticBacteria 16S rRNA and Eukaryotic
18S rRNA – Similar!18S rRNA – Similar!

78. 16S rRNA conservation (red)16S rRNA conservation (red)

79. Gene Regulation

81. Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin
modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription

82. Gene Regulation Example 1
Activators, Enhancers and
Transcription Factors

83. Fig. 18-8-1
Enhancer
(distal control elements)
Proximal
control elements
Poly-A signal
sequence
Termination
region
Downstream
Promoter
Upstream
DNA
ExonExon ExonIntron Intron

84. Fig. 18-8-2
Enhancer
(distal control elements)
Proximal
control elements
Poly-A signal
sequence
Termination
region
Downstream
Promoter
Upstream
DNA
Exon Exon ExonIntronIntron
Cleaved 3′ end
of primary
transcript
Primary RNA
transcript
Poly-A
signal
Transcription
5′
ExonExon ExonIntron Intron

85. Fig. 18-8-3
Enhancer
(distal control elements)
Proximal
control elements
Poly-A signal
sequence
Termination
region
Downstream
Promoter
Upstream
DNA
ExonExon ExonIntron Intron
Exon Exon ExonIntronIntron
Cleaved 3′ end
of primary
transcript
Primary RNA
transcript
Poly-A
signal
Transcription
5′
RNA processing
Intron RNA
Coding segment
mRNA
5′ Cap 5′ UTR
Start
codon
Stop
codon 3′ UTR Poly-A
tail
3′

86. Fig. 18-9-1
Enhancer TATA
box
PromoterActivators
DNA
Gene
Distal control
element

87. Fig. 18-9-2
Enhancer TATA
box
PromoterActivators
DNA
Gene
Distal control
element
Group of
mediator proteins
DNA-bending
protein
General
transcription
factors

88. Fig. 18-9-3
Enhancer TATA
box
PromoterActivators
DNA
Gene
Distal control
element
Group of
mediator proteins
DNA-bending
protein
General
transcription
factors
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex RNA synthesis

89. Fig. 18-10
Control
elements
Enhancer
Available
activators
Albumin gene
(b) Lens cell
Crystallin gene
expressed
Available
activators
LENS CELL
NUCLEUS
LIVER CELL
NUCLEUS
Crystallin gene
Promoter
(a) Liver cell
Crystallin gene
not expressed
Albumin gene
expressed
Albumin gene
not expressed

90. Gene Regulation Example 2
The Operon

91. Fig. 18-2
Regulation
of gene
expression
trpE gene
trpD gene
trpC gene
trpB gene
trpA gene
(b) Regulation of enzyme
production
(a) Regulation of enzyme
activity
Enzyme 1
Enzyme 2
Enzyme 3
Tryptophan
Precursor
Feedback
inhibition

92. Fig. 18-3a
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
mRNA 5′
Protein Inactive
repressor
RNA
polymerase
Regulatory
gene
Promoter Promoter
trp operon
Genes of operon
Operator
Stop codonStart codon
mRNA
trpA
5′
3′
trpR trpE trpD trpC trpB
ABCDE

93. Fig. 18-3b-1
(b) Tryptophan present, repressor active, operon off
Tryptophan
(corepressor)
No RNA made
Active
repressor
mRNA
Protein
DNA

94. Fig. 18-3b-2
(b) Tryptophan present, repressor active, operon off
Tryptophan
(corepressor)
No RNA made
Active
repressor
mRNA
Protein
DNA

95. Fig. 18-4a
(a) Lactose absent, repressor active, operon off
DNA
Protein
Active
repressor
RNA
polymerase
Regulatory
gene
Promoter
Operato
r
mRNA
5′
3′
No
RNA
made
lacI lacZ

96. Fig. 18-4b
(b) Lactose present, repressor inactive, operon on
mRNA
Protein
DNA
mRNA 5′
Inactive
repressor
Allolactose
(inducer)
5′
3′
RNA
polymerase
Permease Transacetylase
lac operon
β-
Galactosidase
lacYlacZ lacAlacI

97. Fig. 18-5
(b) Lactose present, glucose present (cAMP level
low): little lac mRNA synthesized
cAMP
DNA
Inactive lac
repressor
Allolactose
Inactive
CAP
lacI
CAP-binding site
Promoter
Active
CAP
Operator
lacZ
RNA
polymerase
binds and
transcribes
Inactive lac
repressor
lacZ
OperatorPromoter
DNA
CAP-binding site
lacI
RNA
polymerase less
likely to bind
Inactive
CAP
(a) Lactose present, glucose scarce (cAMP level
high): abundant lac mRNA synthesized

98. Gene Regulation Example 3
Epigenetics

99. Epigenetics

100. Epigenetics Intro
http://learn.genetics.utah.edu/content/epigenetics/intro/

103. Utah Epigenetics
http://
learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome

104. Gene Regulation Example 4
RNAi

105. RNAi

107. RNA
Induced
Silencing
Complex

110. Vascular Endothelial Growth Factor

111. Transformation – Recombinant
Organisms

112. Cloning Technologies

113. Fig. 20-4-1
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE

114. Fig. 20-4-2
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant
plasmid

115. Fig. 20-4-3
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant
plasmid
Bacteria carrying
plasmids

116. Fig. 20-4-4
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant
plasmid
Bacteria carrying
plasmids
RESULTS
Colony carrying non-
recombinant plasmid
with intact lacZ gene
One of many
bacterial
clones
Colony carrying recombinant
plasmid with disrupted lacZ gene

118. mtDNA
Theories, Molecular Basis
and Real-World Application

121. “The Other Genome”
mtDNA

122. Endosymbiotic Theory

130. DNA Laboratory at
Milton Academy
• Isolate DNA
from cheek cells.
• Polymerase
Chair Reaction
• Electrophoresis
• Sequence DNA

131. mtDNA Control Region

133. Polymerase Chain Reaction

134. PCR
http://www.dnalc.org/resources/spotlight/index.html

136. Taq DNA Polymerase

137. Fig. 20-8a
5′
Genomic DNA
TECHNIQUE
Target
sequence
3′
3′ 5′

138. Fig. 20-8b
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Primers
New
nucleo-
tides
3′ 5′
3
2
5′ 3′1

139. Fig. 20-8c
Cycle 2
yields
4
molecules

140. Fig. 20-8d
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

141. http://www.youtube.com/watch?v=CQEaX3MiDow
http://www.youtube.com/watch?v=x5yPkxCLads&feature=related

142. Gel Electrophoresis

143. DNA Sequencing

144. Chain Termination Methods
Sanger Methods

145. Dye-terminator sequencing

146. Fig. 20-12
DNA
(template strand)
TECHNIQUE
RESULTS
DNA (template
strand)
DNA
polymerase
Primer Deoxyribonucleotides
Shortest
Dideoxyribonucleotides
(fluorescently tagged)
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Direction
of movement
of strands
Detector
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP

147. Fig. 20-12a
DNA
(template strand)
TECHNIQUE
DNA
polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP

148. Fig. 20-12b
TECHNIQUE
RESULTS
DNA (template
strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Direction
of movement
of strands
Detector
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand

149. Trace File

150. Amplification and clonal
selection

151. Kate Bator
Connor Johnson

152. High-throughput sequencing
Next-Gen Sequencing

153. mtDNA Sequence
http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html