Download - Honors - Dna 1112
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Molecular Biology
Honors Biology
Edgar
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DNA Replication
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Exonuclease
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Fig. 16-UN5
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Fig. 16-13
Topoisomerase
Helicase
Primase Single-strand binding
proteins
RNA
primer
5 5
5 3
3
3
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Fig. 16-16b6
Template
strand
5
5 3
3
RNA primer 3 5
5
3
1
1
3
3
5
5
Okazaki
fragment
1 2
3
3
5
5
1 2
3
3
5
5
1 2
5
5
3
3
Overall direction of replication
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Fig. 16-16a
Overview
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions of replication
1 2
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Helicase
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Topoisomerase and Helicase
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Fig. 20-3-1 Restriction site
DNA
Sticky end
Restriction enzyme cuts sugar-phosphate backbones.
5 3
3 5
1
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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
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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
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Fig. 20-9a
Mixture of DNA mol- ecules of different sizes
Power source
Longer molecules
Shorter molecules
Gel
Anode Cathode
TECHNIQUE
1
2
Power source
– +
+ –
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Fig. 20-9b
RESULTS
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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 -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp 175 bp
DdeI DdeI
DdeI DdeI DdeI DdeI
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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.
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Transcription and Translation
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Gene Regulation
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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
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Fig. 18-8-1
Enhancer
(distal control elements) Proximal
control elements
Poly-A signal sequence
Termination region
Downstream Promoter
Upstream DNA
Exon Exon Exon Intron Intron
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Fig. 18-8-2
Enhancer
(distal control elements) Proximal
control elements
Poly-A signal sequence
Termination region
Downstream Promoter
Upstream DNA
Exon Exon Exon Intron Intron Cleaved 3 end of primary transcript
Primary RNA transcript
Poly-A signal
Transcription
5
Exon Exon Exon Intron Intron
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Fig. 18-8-3
Enhancer
(distal control elements) Proximal
control elements
Poly-A signal sequence
Termination region
Downstream Promoter
Upstream DNA
Exon Exon Exon Intron Intron
Exon Exon Exon Intron Intron 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
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Fig. 18-9-1
Enhancer TATA box
Promoter Activators
DNA Gene
Distal control element
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Fig. 18-9-2
Enhancer TATA box
Promoter Activators
DNA Gene
Distal control element
Group of mediator proteins
DNA-bending
protein
General transcription factors
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Fig. 18-9-3
Enhancer TATA box
Promoter Activators
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
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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
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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
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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 codon Start codon
mRNA
trpA
5
3
trpR trpE trpD trpC trpB
A B C D E
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Fig. 18-3b-1
(b) Tryptophan present, repressor active, operon off
Tryptophan (corepressor)
No RNA made
Active repressor
mRNA
Protein
DNA
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Fig. 18-3b-2
(b) Tryptophan present, repressor active, operon off
Tryptophan (corepressor)
No RNA made
Active repressor
mRNA
Protein
DNA
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Fig. 18-4a
(a) Lactose absent, repressor active, operon off
DNA
Protein Active repressor
RNA polymerase
Regulatory
gene
Promoter
Operator
mRNA 5
3
No RNA made
lacI lacZ
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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
-Galactosidase
lacY lacZ lacA lacI
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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
Operator Promoter
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
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Epigenetics
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Epigenetics Intro
http://learn.genetics.utah.edu/content/epige
netics/intro/
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Genomic
Imprinting
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RNAi
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RNA
Induced
Silencing
Complex
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Vascular Endothelial Growth Factor
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Genetic Engineering & Cloning
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Cloning
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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
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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
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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
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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
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mtDNA
Theories, Molecular Basis
and Real-World Application
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“The Other Genome”
mtDNA
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Endosymbiotic Theory
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DNA Laboratory at
Milton Academy • Isolate DNA
from cheek cells.
• Polymerase
Chair Reaction
• Electrophoresis
• Sequence DNA
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mtDNA Control Region
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Polymerase Chain Reaction
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PCR
http://www.dnalc.org/resources/spotlight/index.html
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Taq DNA Polymerase
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Fig. 20-8a
5
Genomic DNA
TECHNIQUE
Target
sequence
3
3 5
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Fig. 20-8b
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Primers
New
nucleo-
tides
3 5
3
2
5 3 1
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Fig. 20-8c
Cycle 2
yields
4
molecules
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Fig. 20-8d
Cycle 3 yields 8
molecules; 2 molecules
(in white boxes)
match target sequence
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http://www.youtube.com/watch?v=CQEaX3MiDow
http://www.youtube.com/watch?v=x5yPkxCLads&feature=related
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Gel Electrophoresis
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DNA Sequencing
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Chain Termination Methods
Sanger Methods
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Dye-terminator sequencing
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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
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Fig. 20-12a
DNA (template strand)
TECHNIQUE
DNA polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
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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
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Trace File
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Amplification and clonal
selection
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Kate Bator
Connor Johnson
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High-throughput sequencing
Next-Gen Sequencing
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mtDNA Sequence
http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html