Download Week 10 Pre-Lecture Slides

Monday March 6th,
2017
Class 35 Learning Goals
Biotechnology: PCR
• After this class, you should be able to:
– Diagram any stage of a PCR reaction
– Predict the next stage in a PCR reaction and explain the types
and sizes of molecules present
– Describe the process of designing PCR primers
– Understand the process of PCR enough to hypothesize
uses for PCR in analyzing different kinds of DNA-related
issues
Amplifying DNA: The need for many copies
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In chemistry, you’ve use purified solutions of a single
molecule to analyze the characterize that molecule
We need to produce many copies of a single DNA region
The Polymerase Chain Reaction is a method for making many
copies of a single, specific region from any larger DNA
This is called ‘amplification’
Peer Instruction
Explain how this reaction is limited to a
single location in the genome.
Design a DNA primers matching these regions
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3
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Region of DNA to
be amplified by PCR
When target DNA is single-stranded,
-primers bind
-create an accessible 3’OH
-allow DNA polymerase to bind and polymerize
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3
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Primer
Primer
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PCR: Resetting the cycle
Primers
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dNTPs
Temperatures for each step:
1) Denaturation: 90-96C
2) Annealing: 50-60C
3) Extension: 67-72C
PCR: Extending , Cycling
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Explain “Gel Electrophoresis”
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Peer Instruction
A mixture of DNAs
What charge do pieces of DNA have?
In what direction does DNA move through this electrified gel?
Why do shorter fragments move farther through the gel?
Tuesday March 7th,
2017
Class 36 Learning Goals
Biotechnology: Genetic Modification
• After this class, you should be able to:
– Describe each step needed for a human immune gene therapy
procedure.
– Compare and contrast
• human gene therapy with retroviruses,
• plant genetic modification with agrobacterium, and
• DNA modification with CRISPR/Cas.
– Analyze a gene therapy scheme and point out logistically
difficult parts of the procedure
Cloning: Copying a biological entity by an artificial
method
In this case*, cloning is the copying of a gene sequence
for insertion into another genome.
*This is different than cloning an entire organism from a cell.
Cloning: Reverse Transcriptase
Central Dogma:
DNA
RNA
Protein
Reverse Transcriptase:
(developed from viruses)
Single-stranded “cDNA”
Double-stranded cDNA
Primer
Reverse
transcriptase
mRNA
DNA
polymerase
Restriction endonucleases
BamH1 is an enzyme found in a strain of the
bacteria Bacillus amyloliquefaciens.
Peer Instruction
DNA
This enzyme can cut DNA as shown here:
The sequence GGATCC is a palindrome that
Is not found in the Bacillus genome.
1) How do bacteria naturally use the enzyme to destroy viruses?
BamH1 is one of thousands of different
“restriction endonucleases”.
Cut sites for different REs
2) How would a human biologist use a RE?
3) Why is a ‘sticky end’ more useful than
a typical double-stranded break?
DNA of E. coli
Cloning: Plasmid Insertion
To carry and keep this gene safe,
we’ll load it onto a circular DNA plasmid
Recognition site
Recognition site
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Restriction
endonuclease
(EcoR1)
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Plasmid
Sticky end
Transformation: Plasmid Vector
Plasmid
DNA ligase catalyzes a
phosphodiester bond
Recombinant
plasmid
E. Coli cells
Recombinant
plasmid
What is happening in this reaction?
Peer Instruction
Why is a human retrovirus involved?
Viral
RNA
Human
RNA
DNA complementary
to introduced RNA
Double-stranded DNA of
introduced genes
Human cell
Host DNA
Reverse
transcriptase
Peer Instruction
This patient has two defective copies of an
immune-related gene that works in B-cells.
1) What are his doctors attempting?
2) What are these operations like for the patient?
3) Can they prevent the virus from inserting in
important regions of B-cell DNA?
SCID
patient
A plant vector…
Peer Instruction
Agrobacterium cell
Ti
plasmid
Genes that
help insert
the T-DNA
Main chromosome
T-DNA
Genes and promoters that increase cell growth
Host-cell
chromosomes
How does the Agrobacterium make
a safe structure for itself in its host?
Plant cell
nucleus
Inserted T-DNA
Agrobacterium
cells
Explain vector preparation in Agrobacterium.
Tumor-inducing genes
1. Start with normal
T-DNA
Ti plasmids
2. Remove tumorT-DNA
inducing genes.
3. Add genes for
Genes for three
enzymes
enzymes required for carotene synthesis
along with promoter
that will be activated in
endosperm.
Peer Instruction
Peer Instruction
Binds to the Cas9 enzyme
A guide RNA:
Complementary to a genomic sequence
The gRNA enters the nucleus…
Cas9 is guided to the
site and cuts the DNA
The Cas9 enzyme:
• Naturally occurring in bacteria
• Can be transfected into any cell
What can the Cas9 enzyme
do that vectors cannot?
Peer Instruction
Binds to the Cas9 enzyme
A guide RNA:
Complementary to a genomic sequence
The gRNA enters the nucleus…
Break a particular
sequence
Cas9 is guided to the
site and cuts the DNA
Add a dsDNA for
insertion/replacement
Bring a regulating
factor to the site
Wednesday, March 8th,
2017
Class 37 Learning Goals
Genomics
• After this class, you should be able to:
– Assess the likelihood of finding a particular kind of gene in a
human genomic region
– Describe comparative features of several genomes
– Build a small gene map from recombination data
– Describe the differences between a genome, an enterome,
a proteome, a transcriptome, and a biome.
Crossing over in Meiosis
Gene 1
Gene 2
Crossing over is
rare between
genes that are
close together
Gene 1
Crossing over
occurs frequently
between genes
that are far apart
Gene 3
Mapping a chromosome: recombination rates
radi
ceto
drckl
abo
elbow
Forty.2
radi
0
50
19
18
17
4
ceto
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0
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50
50
50
drckl
19
50
0
37
2
15
abo
18
50
37
0
35
22
elbow
17
50
2
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0
13
Forty.2
4
50
15
22
13
0
How to start?
On a chromosome, elbow and forty.2 are separated by 13 distance units.
Radi is 4 units away from forty.2, so it must be in one of two places…
Genetic Mapping: Huntington’s
Disease
Close physical association between
recognition site and defective allele.
Genetic marker
restriction sites
absent
Chromosome of diseased
individual
Chromosome of healthy
individual
Defective
Huntington’s gene
(disease allele)
Normal
Huntington’s gene
Genetic marker
restriction sites
present
Genetic markers at
other locations are
equally likely to be
found in affected and
unaffected individuals
Peer Instruction
HD brain:
What causes Huntington’s disease?
Normal
Htt protein
Wt brain:
Mutant
Htt protein
Normal allele:
ATGCGCGTGATAGCTGATAGCGAGCAG[26xCAG]CAGTTAGCGATTA…
M R V I A E S D Q
26xQ
Q L A I…
Disease causing allele:
ATGCGCGTGATAGCTGATAGCGAGCAG[150+ x CAG]CAGTTAGCGATTA…
M R V I A E S D Q
150xQ
Q L A I…
First genome sequenced: a 3,500 bp RNA phage ‘76
First DNA genome: a 5,000 bp DNA phage in ‘77
First prokaryote: A bacteria in ‘95 (1.8 million bp)
First eukaryote: A yeast in ‘96 (12 million bp)
First animal: C. elegans worm in ‘96 (100 million)
Human genome “finished”: ‘00
(~70% of 3.2 billion bp…currently ~93%)
Current # of genomes: 180+ and growing
Year
Billions of nucleotides sequenced
Genome Sequencing
Sequencing:
Two methods: Slow and Shotgun
primer
new sequence
known sequence
~160-kb fragments
1. Cut DNA at random locations
into fragments of ~160 kb.
Genomic DNA
BAC library
1-kb fragments
“Shotgun
clones”
Shotgun
sequences
BAC
Main bacterial
chromosome
Many copies (three shown)
of each 160-kb fragment,
each cut differently
2. Clone using BACs.
3. Cut into 1-kb fragments.
4. Clone using plasmids.
5. Sequence each fragment.
6. Assemble all the 1-kb fragments.
Draft sequence
7. Assemble all the 160-kb fragments.
Peer Instruction
This is data from the human genome.
What do you notice?
Repeated DNA (centromeres, telomeres)
Genes
‘Junk’ DNA
Mobile genetic elements like
transposons (both viable and ‘dead’)
What do you notice about
that 5% labeled ‘genes’?
Peer Instruction
Protein trafficking
Cell cycle
Cell proliferation and differentiati
Cell structure and motility
Transport
Immunity and defense
Unknown
Developmental
processes
Other metabolism
Misc.
function
Signal
transduction
Protein metabolism
and modification
Nucleoside, nucleotide,
and nucleic acid metabolism
Peer Instruction
Genome size (Mbp)
What does this data tell us?
Nonparasitic
bacteria
Parasitic bacteria
Number of protein-encoding genes
Can you find a trend in genome size and number of genes?
What would be most closely related to a species with 220
million base pairs and ~27,000 genes?
Developmental
processes
Protocadherins are the largest subgroup
within the cadherin family of Ca-dependent
cell-cell adhesion molecules. Interestingly, many of
the protocadherins in mammals are highly expressed
in the central nervous system. In the postnatal
mammal brain, protocadherins are possibly involved
in the modulation of synaptic transmission and the
generation of specific synaptic connections.
Human
Octopus
Total amount of DNA
3.1 gbp
2.7 gbp
Total # of genes
~25,000
<33,000
Total # of
protocadherin genes
58
168
What happened in octopus evolution?
Unique arm neurons in this mollusk allow highly coordinated creative
sensory and motor action…even to act independently. This distributed
neural network makes octopi uniquely intelligent among invertebrates.
Here is a species with 220 million base pairs and ~27,000 genes:
Tetrahymena:
• Unicellular
• Ciliate
• Rotifer
bacterial prey
Gene duplication and evolution
8 repeats
1. Homologs pair up.
8 repeats
2. Repeats misalign. Crossing
over and recombination occur.
Chromosomes break
and exchange here
10 repeats
3. Products are unique.
6 repeats
Globin gene family
Pseudogene
Coding gene
Proteome research
• The set of all proteins in a cell is the
‘proteome’
• Because protein is the functional unit in
many systems, analyzing protein levels
can give exact data
– Can decipher alternative splicing
variations
– Can account for post-translational
modification and degradation
– Allows for time-sensitive analysis of
signals and responses
– However, it is very difficult due to the
variety of structures possible
• Mass spectrometry can help analyze
cell protein content
• Large-scale binding searches
Transcriptome Research: Microarrays
Normal
temperature
High
temperature
Example of a
functional genomics
comparison
1. Isolate mRNAs
and use reverse
transcriptase to
prepare singlestranded cDNA.
Reverse
transcriptase
mRNA
cDNA
cDNA
probes
2. Make cDNA
probes.
Microarrays:
Visualizing the
data
3. Probe a
microarray.
Microarray
Microarray computer output:
4. Shine laser light
on one spot at a
time to induce
fluorescence.
Each gene transcribed
in the experimental
condition is a candidate
for further research…
Green spots: Yellow spots:
genes
genes
transcribed
transcribed
at normal
equally in
temperature
both cells
Dark spots:
low gene
expression
Red spots:
genes
transcribed
at high
temperature
Thursday March 9th,
2017
Class 37 Learning Goals
Antibiotics and the Human Microbiome
• After this class, you should be able to:
– Express antibiotics as a temporary and limited means of
controlling rapidly evolving bacteria
– Describe multiple differences between similar individuals in
terms of their microbiota
– Hypothesize causes or potential treatments for human diseases
based on microbiomal disruptions
Peer Instruction
What do these data
tell us about human
health care?
In your body:
Human cells: somewhere between 20-40 trillion
Human cells: only about 50% of the total…
What is a microbiome?
What is an enterotype?
…Here
Peer Instruction
is the other 50%
Peer Instruction
How can microbiomes differ between people?
What are the differences
between young and old people?
What are the overall differences
between these ethic group?
Peer Instruction
What would be the most efficient way to donate
fecal microbiota from one person to another?
Hint:
How could you use microbiome information to tell who
has been in a room or not?
Why should new parents create skin to skin contact with babies?
Why are researchers interested in gastric cancer starting to focus
on the enteromes of their patients?