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Fig. 6-1
What is in our genome?
Ge
ne
s
A large fraction of our genome does not produce
gene products (with known function)!
Simple-sequence - repetitious DNA
(≈6% of human genome)
Satellite DNA: 14-500 bp repeats in tandem, 20-
100kb (Heterochromatin – telomeres and
centromeres)
Microsatellites: 1-13bp repeats in tandem, up to
150bp
How do these
repeats arise? Fig 6-39
Figure 21.7
1. Homologs pair up.
2. Repeats misalign.
Crossing over and
recombination occur.
3. New repeat
numbers are created.
8 repeats
8 repeats
10 repeats
6 repeats
Chromosomes break
and exchange here
The number of repeats can change through unequal
crossing over during meiosis
Result: # repeats can be highly
variable between individuals - useful for DNA fingerprinting or
disease diagnosis Biology (Campbell, 9 ed) Fig 13-12
Meiosis I:
Meiosis II:
n - 1
Simple-sequence repeats may occur through
replication errors
Fig 6-5
The PCR method is used to amplify DNA sequences
– Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule
– Relies upon a pair of primers – Short DNA molecules that bind to sequences at each end
of the sequence to be copied
– Used as a starting point for DNA replication
Copyright © 2009 Pearson Education, Inc.
Fig 5-20
heat, 95˚C
cool, 50-72˚C
72˚C
DNA template
Primer Primer
5’
5’ 3’
3’ 3’ 5’ 5’ 3’
Basics of PCR (polymerase chain reaction)
WHAT IS NEEDED FOR A PCR REACTION?
Exponential amplification of DNA – up to 2(#cycles)
30 cycles => up to 230 ≈ 109 fold amplification Fig 5-20
Basics of PCR (polymerase chain reaction)
Using PCR to detect repeat expansions
Primer 1
Primer 2
Primer 1
Primer 2
Amplification by PCR
Amplification by PCR
Smaller PCR products
Larger PCR products
Person 1
Person 2
Gel electrophoresis sorts DNA molecules by size
– Gel electrophoresis separates DNA molecules based on size
– DNA sample is placed at one end of a porous gel
– Current is applied and DNA molecules move from the negative electrode toward the positive electrode
– WHY?
Copyright © 2009 Pearson Education, Inc.
DNA ladders or standards
• Whenever we run an agarose gel, DNA standards of known size are run as a guide to determine the molecular weight or amount of the DNA fragments in the experimental samples. These standards are often referred to as “ladders”.
• The standard we will using consists of the genome from lambda bacteriophage digested with the enzyme Hindlll.
• The resulting 7 fragments are 564, 2027, 2322, 4361, 6557, 9416, 23130 bp in size.
– (A 125 bp fragment is present but usually not seen.)
1 2 3
23,130 bp
9,416 bp
6,557 bp
4,361bp
2,322 bp 2,027 bp
Lane 1: lambda HIND lll standard Lane 2: unknown X Lane 3: unknown Y
We will using an agarose gel to determine both the size of the DNA
fragments in an unknown sample…
To determine size, compare how far bands have migrated relative to the standards – how big are the DNA molecules in sample X and Y?
We know this band contains 240 ng of DNA (assuming you loaded 10 ml of Hindlll lambda standard)
How much DNA would you estimate is in this lane in comparison to standard band?
Take that number and divide by amount of DNA you loaded onto gel = concentration of DNA in your sample.
Note: you divide by the total amount of DNA you loaded, not including the sample buffer or any water.
…and to get an idea of the amount of DNA in our samples by
comparing the intensity of the bands to those in the standards
DNA fingerprinting
PCR on multiple variable repeat
regions (several different
primer pairs) +
Gel electrophoresis
Fig 6-7
Who is the father?
A)F1
B)F2
DNA fingerprinting
PCR on multiple variable repeat
regions (several different
primer pairs) +
Gel electrophoresis
Fig 6-7
Who is guilty?
A)1 B)2 C)3
Table 16.1 Genomes 3 (© Garland Science 2007)
Diseases associated with repeat expansions
PCR can be used as diagnostic test for disease
• Huntington’s Disease: neurodegenerative disease caused by a polyglutamine expansion in the HTT protein (VIDEO)
Below is shown a region of genomic DNA containing repeat sequences.
You wish to generate the PCR product shown below.
Genomic DNA:
5’...CTGCGCGGCAGGTTGTAGTCAGCAGCAGCAGTGCGTATTTGAGTAGA...3’
3’...GACGCGCCGTCCAACATCAGTCGTCGTCGTCACGCATAAACTCATCT...5’
A: 5’-TCCAACATCA-3’ and 5’-AAATACGCA-3’
B: 5’-AGGTTGTAGT-3’ and 5’-ACGCATAAA-3’
C: 5’-ACTACAACCT-3’ and 5’-TGCGTATTT-3’
D: 5’-AGGTTGTAGT-3’ and 5’-AAATACGCA-3’
E: 5’-TCCAACATCA-3’ and 5’-TGCGTATTT-3’
Desired PCR Product:
5’-AGGTTGTAGTCAGCAGCAGCAGTGCGTATTT-3’
3’-TCCAACATCAGTCGTCGTCGTCACGCATAAA-5’
Which primers would you use?
Repeat
Below is shown a region of genomic DNA containing repeat sequences.
You wish to generate the PCR product shown below.
Genomic DNA:
5’...CTGCGCGGCAGGTTGTAGTCAGCAGCAGCAGTGCGTATTTGAGTAGA...3’
3’...GACGCGCCGTCCAACATCAGTCGTCGTCGTCACGCATAAACTCATCT...5’
A: 5’-TCCAACATCA-3’ and 5’-AAATACGCA-3’
B: 5’-AGGTTGTAGT-3’ and 5’-ACGCATAAA-3’
C: 5’-ACTACAACCT-3’ and 5’-TGCGTATTT-3’
D: 5’-AGGTTGTAGT-3’ and 5’-AAATACGCA-3’
E: 5’-TCCAACATCA-3’ and 5’-TGCGTATTT-3’
Desired PCR Product:
5’-AGGTTGTAGTCAGCAGCAGCAGTGCGTATTT-3’
3’-TCCAACATCAGTCGTCGTCGTCACGCATAAA-5’
Which primers would you use?
Repeat
More than 3 million mobile elements in our genome!
(~45%)
Ge
ne
s
Mobile DNA elements = Transposable elements
DNA can move within and between chromosomes ~1940s – very controversial idea!
VIDEO
Barbara McClintock MacArthur “Genius” Award, 1981
Lasker Award, 1981
Nobel Prize, 1983
Summer of 1944: Cold Spring Harbor Labs, Long Island
Developed the transposon theory by studying corn kernel coloration.
Wild-Type
Mutant Revertants
Two general types of transposition
Fig 6-8
Cut&Paste Copy&Paste
DNA transposons Retrotransposons
Bacterial DNA transposons –
IS (insertion sequences)
Transposase
Fig. 10-9
Fig 6-9
Encodes
Catalyses DNA transposition
Transposition of a bacterial IS transposon
Fig 6-10
Encoded by Transposon
Cellular DNA repair enzymes
Cut target DNA
Cut Transposon
Creates target site direct repeats
You add a drug that inhibits cellular
DNA Ligase to a cell undergoing IS
element DNA transposition.
According to the model for DNA
transposition, which Target DNA
intermediate of the transposition
reaction should accumulate?
5’ 5’ 3’ 3’
3’ 3’ 5’ 5’
5’ 3’
3’ 5’
5’
3’
3’
5’
A:
B:
C:
D:
E:
DNA Transposition reaction
You add a drug that inhibits cellular
DNA Ligase to a cell undergoing IS
element DNA transposition.
According to the model for DNA
transposition, which Target DNA
intermediate of the transposition
reaction should accumulate?
5’ 5’ 3’ 3’
3’ 3’ 5’ 5’
5’ 3’
3’ 5’
5’
3’
3’
5’
A:
B:
C:
D:
E:
DNA Transposition reaction
DNA transposons can increase in copy number
during DNA replication
Eukaryotic retrotransposons
Reverse Transcriptase
Fig 6-12
LTR: Long terminal repeat
NOTE: Similar to Retroviruses – but lack envelope proteins
Integrase
and
Encodes
Catalyze Retro-transposition
General mechanism of Retrotransposition
Based on Fig 6-12
Retrotransposon RNA
Retrotransposon DNA
Target DNA
1) Reverse Transcriptase makes DNA
copy
Encoded by Transposon
2) Integrase – inserts Retrotransposon
into target DNA
Fig 6-16
LINEs: - Long Interspersed Elements
- 21% of total human DNA (900,000!)
- ~6,000bp intact, but 99.99% are not
SINEs: - Short Interspersed Elements (Lack ORFs)
(a class of SINEs = Alu elements)
- rely on LINE protein for retrotransposition
- ~300bp
- ~13% of total human DNA (1,600,000!)
Common types of human retrotransposons
Alu Elements
• Over 1 million in genome
• 10.7% of genome
• Connected to many cancers, diabetes, hemophilia, etc.
• Can be used to trace human history
A: It is a DNA transposon.
B: It is a retrotransposon.
C: It could be either a DNA transposon
or a retrotransposon.
D: It is neither a DNA transposon or a
retrotransposon
You are studying a new mobile element in yeast, and want to determine if it is a
retrotransposon or a DNA transposon.
To do so, you perform an experiment: You treat one culture of growing yeast
with an RNA polymerase inhibitor while leaving a second culture untreated.
The next day, you find that the mobile element has transposed to new target
sites in the untreated culture, but did not in the treated culture.
What can you conclude from these data about the
mobile element?
RNA pol inhibitor
added untreated
transposition occurred
transposition blocked
A: It is a DNA transposon.
B: It is a retrotransposon.
C: It could be either a DNA transposon
or a retrotransposon.
D: It is neither a DNA transposon or a
retrotransposon
You are studying a new mobile element in yeast, and want to determine if it is a
retrotransposon or a DNA transposon.
To do so, you perform an experiment: You treat one culture of growing yeast
with an RNA polymerase inhibitor while leaving a second culture untreated.
The next day, you find that the mobile element has transposed to new target
sites in the untreated culture, but did not in the treated culture.
What can you conclude from these data about the
mobile element?
RNA pol inhibitor
added untreated
transposition occurred
transposition blocked
Transposons are kept (mostly) silent
Strong silencing maintained at both the transcriptional and post
transcriptional levels: chromatin structure and RNAi (more on
this in future weeks)
When would it be
particularly important to
CONTROL transpositions?
1. Generation of gene families – duplications through homologous sites
for unequal crossing over
2. Creation of new genes - exon shuffling
3. Formation of complex regulatory regions that control gene expression
Mobile DNA elements and genome evolution
25% of our genome is “unclassified”!
Spacer DNA – really “junk”??
Role in gene expression? • complex transcriptional control regions affecting promoter activity
Role in cellular organization? • may impact structure of chromosomes and how DNA is organized
in the nucleus
Unknown genes - new proteins or non-coding RNAs? • Deep sequencing of RNA transcripts = lots of transcription
occurring in spacer DNA!
Provide space between genes to isolate the effect of mutation.
How can we study the function of a gene?
Gene cloning Allows study of the gene in separation from the remainder of the genome. Gene inactivation Allows study of the cellular/organismal effect of loss of gene function.
How do you isolate and propagate a piece of DNA (for example a gene)?
Genomic or Viral DNA
Vector (= plasmid or viral DNA that can replicate in a desired
organism - often E. coli)
Cut out piece of DNA
Insert into vector Amplify in
cells
(e.g. E. coli)
GAATTC
CTTAAG
5’
5’
3’
3’
G 3’ 5’ AATTC
CTTAA 5’ 3’ G
5’
5’
3’
3’
GGTACC
CCATGG
5’
5’
3’
3’
GGTAC 3’ 5’ C
C 5’ 3’ CATGG
5’
5’
3’
3’
Restriction enzymes cleave DNA at specific (usually palindromic) sequences
EcoRI
KpnI
Similar to Fig. 5-11
Similar to Table 5-1
Based on Fig. 4-1
Many bacteria contain restriction-modification systems to
“restrict” invasion by foreign DNA
GAATTC
CTTAAG
CH3
I
I
CH3
Bacterial genome DNA Invading DNA (e.g. virus)
GAATTC
CTTAAG
5’
5’
3’
3’
G 3’ 5’ AATTC
CTTAA 5’ 3’ G
5’
5’
3’
3’
5’
3’ 5’
3’
Modifying enzyme (methylase)
EcoRI
EcoRI
Where do restriction enzymes come from?
Fig. 5-12
Ligating a DNA fragment to a vector
Vector
DNA fragment
Obtaining bacterial clones with your recombinant plasmid
Fig. 5-14
Bacterial colonies containing your plasmid (only plasmid-containing bacteria will survive on
ampicillin)
Pick a colony - you have your
clone!
Plate on selective media (e.g. ampicillin)
What - in addition to a selectable marker gene - is required in
a plasmid to ensure that bacteria retain it through multiple
generations?
A: A telomere
B: An origin of replication
C: A restriction site
D: A gene for a modifying enzyme
What - in addition to a selectable marker gene - is required in
a plasmid to ensure that bacteria retain it through multiple
generations?
A: A telomere
B: An origin of replication
C: A restriction site
D: A gene for a modifying enzyme
How do you obtain a specific insert DNA (for example a gene)?
From a DNA source (e.g. genomic DNA)
- PCR
- Genomic library
From an RNA source (e.g. total cellular RNA)
- RT-PCR to create a cDNA library
Genomic DNA
Creating a genomic DNA insert using PCR
PCR
Ligation into vector
- Transform to bacteria,
- Select
see Fig 5-24
Primer 1
Primer 2
PCR product (e.g. a gene)
Creating a copy DNA (cDNA) clone from mRNA (RT-PCR)
mRNA
Primer 2
Reverse Transcriptase + dNTPs
mRNA
cDNA
5’ 3’ 5’
5’ 3’ 5’
3’
PCR
Primer 1
Primer 2
RNase H
cDNA 3’ 5’
3’ 5’ ds-cDNA 5’ 3’
- Ligate into vector
- Transform to bacteria,
- Select
Similar to Fig 5-15
Restriction sites can be added via primer sequences
PCR
Primer 1
Primer 2
3’ 5’ PCR product 5’ 3’
For example:
EcoRI
BamHI
BamHI EcoRI
DNA
Restriction digest, Ligation into vector
BamHI EcoRI
It is sometimes critical to have a specific orientation of the insert in a vector, for
example when you want to express an inserted gene from a pre-existing promoter
in the plasmid.
What would be the orientation of the insert in the following cloning?
HindIII EcoRI
HindIII EcoRI
Insert Plasmid
HindIII: A AGCTT EcoRI: G AATTC
HindIII EcoRI HindIII EcoRI
A B
C: Mix of ‘A’ and ‘B’
D: Neither ‘A’ nor ‘B’
It is sometimes critical to have a specific orientation of the insert in a vector, for
example when you want to express an inserted gene from a pre-existing promoter
in the plasmid.
What would be the orientation of the insert in the following cloning?
HindIII EcoRI
HindIII EcoRI
Insert Plasmid
HindIII: A AGCTT EcoRI: G AATTC
HindIII EcoRI HindIII EcoRI
A B C: Mix of ‘A’ and ‘B’
D: Neither ‘A’ nor ‘B’
How do you know your bacterial clone contains your plasmid (and not e.g. self-ligated plasmid)?
Pick a colony (and expand in
selective medium)
Isolate plasmid
Restriction digest (e.g. BamHI)
Analyze by agarose gel
electrophoresis
Large DNA
Small DNA
Fig. 5.1
-
+
Stain with DNA-specific dye (e.g. EtBr)
Analysis by restriction digestion
Pick 5 clones (A-E); Restriction digest
isolated plasmids with BamHI;
Separate in agarose gel
4,000 bp
500 bp
4,000 bp
3,000 bp
2,000 bp
1,000 bp
A B C D E
Which clone (A-E) has the correct plasmid?
WHY DO ALL CELLS IN A SINGLE COLONY HAVE THE SAME PLASMID?
Pick 5 clones (A-E); Restriction digest
isolated plasmids with BamHI;
Separate in agarose gel
4,000 bp
500 bp
4,000 bp
3,000 bp
2,000 bp
1,000 bp
A B C D E
Which clone (A-E) has the
correct plasmid?
WHY DO ALL CELLS IN A SINGLE COLONY HAVE THE SAME PLASMID?
6-3
Creating a genomic library
Genomic DNA
Chop up with restriction enzyme
Transform to bacteria, select
- Each clone will have a plasmid with a specific genomic insert.
- Different clones contain different inserts.
Similar to Fig 5-17
Ligate with plasmid
Multiple plasmids, each with different DNA inserts!
DNA fragments
- Ligate to plasmid,
- Transform to bacteria,
- Select (= cDNA library) see Fig 5-15
Creating a eukaryotic cDNA library
mRNAs
oligo-dT Primer
Reverse Transcriptase + dNTPs
mRNA
cDNA
5’ 3’
5’
5’ 3’ 5’
3’
AAAAAAA
AAAAAAA TTTTTTTT
Will anneal to all poly(A) tails!
RNase H (degrades RNA in RNA:DNA hybrid)
RNA oligo + RNA ligase
cDNA 5’ 3’ TTTTTTTT
ds-cDNA 5’ 3’ TTTTTTTT
Primer AAAAAAA
DNA polymerase + primer (annealing to 5’ end)
For example:
Remove cap 5’ 3’ AAAAAAA
Added to all mRNAs!
Finding the colony that contains a plasmid with your gene of interest in a library
32P-labeled probe (DNA or RNA) hybridizing to your gene of interest
Fig 5-16
Colony hybridization
What can you do with your gene clone?
Vector
Gene of interest
- Express protein (for example in bacteria) For studies of protein function/structure. For medical or nutritional production of protein. - Express gene, or mutant gene, in organism of origin For studies of gene expression and/or function. Gene therapy???
How can we study the function of a gene?
Gene cloning Allows study of the gene in separation from the remainder of the genome. Gene inactivation Allows study of the cellular/organismal effect of loss of gene function.
Gene inactivation in yeast
Gives resistance to G-418
Fig 5-39
Same principle used in
bacterial gene inactivation
1) Generate a DNA that contains regions
flanking the gene to be knocked out
2) Recombine the DNA into the genome
Genomic sequence
Conditional gene inactivation in mice using Cre/lox
Fig 5-42
Multiple steps of gene expression
Fig. 1-11
TRANSCRIPTION: Production of a
primary RNA transcript from a gene
The process of transcription
DNA Template strand
5’-to-3’ RNA strand growth
Incoming NTP
3’ 5’
5’ 3’
Based on Fig. 4-10
Base pairing
Release of pyrophosphate (PPi)
RNA Polymerase
Which of these nucleotides is used in Transcription?
(A) (B) (C)
(D)
Which of these nucleotides is used in Transcription?
(A) (B) (C)
(D)
RNA polymerase
Genomic DNA
?????
How does RNA polymerase know where to start transcription?
RNA polymerase
E. coli promoter
RNA polymerases recognize promoter sequences Prokaryote model –
5’ 3’
5’ 3’ DNA
+1
Sigma (s) factor
E. coli promoter
s factor binds conserved promoter elements (In normal E. coli genes: -10, -35)
RNA polymerase (a2bb’s)
E. coli RNA polymerase is a multi-subunit protein complex
+1
a2
b
b
Common eukaryotic RNA Polymerase II promoter elements
Transcription start (+1)
Upstream element: CpG islands upstream of start site
BRE: TFIIB Recognition Element
TATA: TATA box
Inr: Initiator element
DPE: Downstream Promoter Element
Note: Few (if any) Pol-II promoters contain all of these elements! None of these elements found in all Pol-II promoters!
Jim Kadonaga, UCSD
The three steps of transcription
5’
5’
3’
Template strand
Non-template strand RNA polymerase
DNA
RNA
Primary transcript Similar to
Fig. 4-11
Melts the DNA
RNA Pol synthesizes RNA 5’-to-3’
Termination releases RNA Pol and RNA
Bacterial RNA Polymerase
Eukaryotic mRNA
The three steps of transcription
5’
5’
3’
Template strand
Non-template strand RNA polymerase
DNA
RNA
Primary transcript Similar to
Fig. 4-11
Melts the DNA
RNA Pol synthesizes RNA 5’-to-3’
Termination releases RNA Pol and RNA
Fig. 7-8
Biochemical fractionation of eukaryotic nuclei identified three eukaryotic RNA polymerases
Which polymerase transcribes protein-coding genes?
a-amanitin inhibits transcription of protein-coding genes and RNA Polymerase II
Eukaryotic RNA Polymerases specialize in transcribing specific types of genes
Eukaryotic vs. Prokaryotic RNA Polymerases
RNA Polymerases contain multiple subunits
Fig. 7-10
Pol II large subunit C-
terminal domain (CTD):
- Multiple copies (52 in
human) of seven-amino acid
repeat, incl 3 serines.
- CTD phosphorylation is
critical for txn initiation.
RNA polymerase
A. RNA polymerase I
B. RNA polymerase II
C. RNA polymerase III
D. The majority of miRNAs are produced by RNA pol II, a minority by RNA pol III
E. RNA polymerases I, II and III are all used to produce miRNAs
The human genome encodes hundreds of 21-23 nucleotide RNAs that regulate
mRNA translation called miRNAs. You wish to identify the RNA polymerase(s)
responsible for transcription of miRNAs and pulse-32P-label all RNA in cells in the
presence of various concentrations of a-amanitin, isolate miRNAs and subject
them to gel electrophoresis. Based on the result, which is the polymerase used?
miRNAs
A. RNA polymerase I
B. RNA polymerase II
C. RNA polymerase III
D. The majority of miRNAs are produced by RNA pol II, a minority by RNA pol III
E. RNA polymerases I, II and III are all used to produce miRNAs
The human genome encodes hundreds of 21-23 nucleotide RNAs that regulate
mRNA translation called miRNAs. You wish to identify the RNA polymerase(s)
responsible for transcription of miRNAs and pulse-32P-label all RNA in cells in the
presence of various concentrations of a-amanitin, isolate miRNAs and subject
them to gel electrophoresis. Based on the result, which is the polymerase used?
miRNAs
7-3
How do we know the DNA elements that control transcription?
Based on Fig. 7-13
Assay for reporter gene expression
(in vitro or in vivo)
Reporter Assay:
1. Northern blotting (Detects RNA levels using probes).
2. Easily assayed gene products.
For example:
a. X-gal galactose + BLUE
b-galactosidase (LacZ)
b. luciferin light emission
luciferase
c. GFP - protein that fluoresces green under UV light
How can we detect reporter gene expression?
How do you detect a specific cellular RNA?
+
-
1. isolate total RNA
4. Detect using radiolabeled
antisense probe
3. transfer to membrane,
fix, hybridize
2. denature and separate RNA
by electrophoresis
Experimental detection of RNA by Northern blotting
FOR EXAMPLE: Northern blot for b-globin mRNA in differentiating erythrocytes
Hours after differentiation:
Fig. 5-27
1. Northern blotting (Detects RNA levels using probes).
2. Easily assayed gene products.
For example:
a. X-gal galactose + BLUE
b-galactosidase (LacZ)
b. luciferin light emission
luciferase
c. GFP - protein that fluoresces green under UV light
How can we detect reporter gene expression?
Osamu Shimomura
Roger Tsien, UCSD
Nobel Prize
Chemistry 2008
Which of the following 32P-labeled DNA probes could be used
to detect the mRNA shown above?
5’CGGUAAAUGGUUAGUCGAUGGGUUCUCGAUGAGC3’ mRNA:
A: 5’-32P-CCCAAGAGCTACT-3’
B: 5’-32P-GGGTTCTCGATGA-3’
C: 5’-32P-AGTAGCTCTTGGG-3’
D: 5’-32P-TCATCGAGAACCC-3’
Which of the following 32P-labeled DNA probes could be used
to detect the mRNA shown above?
5’CGGUAAAUGGUUAGUCGAUGGGUUCUCGAUGAGC3’ mRNA:
A: 5’-32P-CCCAAGAGCTACT-3’
B: 5’-32P-GGGTTCTCGATGA-3’
C: 5’-32P-AGTAGCTCTTGGG-3’
D: 5’-32P-TCATCGAGAACCC-3’
Which specific promoter
sequences are required for
transcription? Linker scanning
mutations
Reporter mRNA
+1 -100
Unrelated mRNA
WT
Reporter mRNA
+1 -100
Unrelated mRNA
WT
D