MOLECULAR GENETICS
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Transcript MOLECULAR GENETICS
INTRODUCTION TO THE
WAKSMAN RESEARCH
PROJECT
DNA Sequence Analysis
of the Duckweed
Wolffia arrhiza
PROBLEM
What genes are present in
Wolffia arrhiza?
What are the functions of
these genes?
WHY DO WE WANT TO
IDENTIFY GENES AND THEIR
FUNCTIONS?
Identifying genes and their
functions we can cure:
Genetic disorders
abnormal genes making abnormal products-block
abnormal genes not making essential products-replace
Cancer
Identify genes involved-turn on or off
Ongogenes-turn off
Tumor suppressor-genes-turn on
Identifying genes and their
functions we can cure:
Diabetes
Add insulin making gene
Spinal cord injuries
Turn on nerve cell genes for cell division
Infectious diseases
Turn off vital genes-kill organism
Alzheimer's
Turn off genes making abnormal proteins
Why Wolffia arrhiza?
Smallest flowering plant
Grows in slow moving fresh water -world wide
Fast reproduction - doubles in a few days
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Duckweed-The little plant
that can save the world
Potential biofuel source
• Under cold temperatures can accumulate 40%-70% starch
• Sink to bottom of ponds
• Starch can easily be converted to sugar for fermentation
• Will not compete with food crop production
Bioremediation
• Grows in contaminated (polluted) water
• Sequesters or degrades contaminates such as lead,
arsenate, halogenated compounds
• Reduces excess nitrogen and phosphate from waste water
Potential food source
Possible source of inexpensive protein
Reduces global warming and produces oxygen
Duckweed may serve as a
model organism
Model Organisms:
A model
organism is a
species that is
extensively
studied to
understand
biological
phenomena
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Model Organisms:
It is understood
that discoveries
made in model
organism will
provide insight
into the working
of other
organisms
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Model Organisms
Model organisms
are widely used to
explore potential
causes and
treatments for
human diseases
when human
experimentation is
unfeasible
Why model organisms
work?
Model organism
strategy made
possible by the
common descent
of all living
organisms and the
conservation of
metabolic and
developmental
pathways and
genetic material
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Why are we able to apply
knowledge obtained from
model organisms to
humans?
Evolution-similarities among organisms are
based on common ancestries
Universal genetic code
All use same four nucleotide bases
All use same 20 amino acids
All use ATP
All made up of cells
Important genes are
conserved genes
The more essential a gene is the
less likely is to have mutated.
Thus essential genes will be very
similar among organisms
In order to study DNA, it must
be amplified and eventually
purified and stored
To purify a gene means to
isolate gene from rest of
DNA and cell
For many years, biochemists had tried
to purify genes.
But they were frustrated because they are hard to
purify.
Because genes are composed of
A’s, C’s, G’s, and T’s, they all pretty
much are chemically alike.
Also genes are parts of chromosomes.
Chromosomes break easily and
randomly, often in the middle of genes.
So how did scientists
eventually purify
individual genes?
Amplification means to make
many copies of a gene
Once you have many copies
of a purified gene you need a
way to store it for future use
and research
How can you accomplish
purification, amplification and
storage of a gene?
VECTORS ALLOW US TO
ACCOMPLISH ALL THREE
TASKS
What is a vector?
Any vehicle that can carry
DNA into a host cell
Once inside the host cell it has
the ability to replicate itself
and any inserted DNA
Amplification can be accomplished
thru cloning with a vector
What are the three steps in
cloning?
DNA of organism must be broken down
into smaller pieces
Pieces of DNA must be joined to another
piece of DNA (vector) that can replicate
itself and the DNA of interest
Vector plus its joined insert must be
introduced into a living cell (living cells
act as copying machines)
Many types of vectors
YACS-400,000 bp
BACS-100-300 bp
Lambda phage-20,000 bp
Cosmids-40,000 bp
Plasmid-type of vector we are
using
What is a plasmid?
Type of vector
Small, circular, self replicating extra
piece of DNA completely distinct
from chromosomal DNA
Found naturally in bacteria and
yeast
Contains a small number of genes
not required for survival under
normal conditions
What is a Plasmid?
Can give a survival advantage to
bacteria living in a stressed
environment
Example-antibiotic resistance
Due to high energy requirement of
maintaining and replicating
plasmids, only plasmids that confer
an advantage are kept by
organism
What is a Plasmid?
Replicated by the host’s machinery
independently of the genome. This is
accomplished by a sequence on the
plasmid called ori, for origin of
replication.
Some plasmids are present in E. coli
at 200-500 copies/cell
What is a plasmid?
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Two Types of plasmids
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What type of plasmid are
we using?
What are important
properties of pDNR-Lib?
Important feature of
plasmid
Plasmids also contain selectable markers.
Genes encoding proteins which provide a
selection for rapidly and easily finding
bacteria containing the plasmid.
Provide resistance to an antibiotic
(ampicillin, kanamycin, tetracycline,
chloramphenicol, etc.).
Thus, bacteria will grow on medium
containing these antibiotics only if the
bacteria contain a plasmid with the
appropriate selectable marker.
Plasmid Characteristics
Ori
Selectable
Marker
3.6 Kb
Color screen
(not in this
plasmid)
MCS
Circular DNA
Color
Screen-not
found in
pDNR-LIB
but
important
in many
other
types of
plasmids
What tools do we use to cut DNA of interest and
join it to a plasmid?
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Must get plasmid with insert
into host cell
Transformationintroduction of
foreign plasmid
into a bacteria
What are restriction
enzymes?
Cut DNA at defined sequences 4-8
bp long called restriction sites
Cut phosphodiester bonds that link
nucleotides together
Cut in a precise and predictable
manor, thus reproducible
Restriction fragments-piece of cut
DNA
Where do restriction
enzymes come from?
Example-EcoR1 restriction
enzyme
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How do we know their will
be our restriction site
Restriction site sequences
occur randomly many times in
a long DNA molecule
Probability of six base
sequence
46=4,096 bp
How are restriction enzymes
named?
EcoRI from Escherichia
coli
BamHI from Bacillus
amyloliqueraciens
PvuI and PvuII are different
enzymes from same strain.
Genus-species-strain-order of
discovery
What happens if we cut
Duckweed DNA and our
plasmid with EcoR1?
Example on board
Why do we add ligase?
Link together nucleotides
Phosphodiester bonds
Dehydration synthesis
When we work with enzymes
must create optimal working
environment
Need buffer (pH, salt conc)
Proper temperature
Poor conditions may:
deactivate enzyme
cause starr activity
What restriction enzymes do
we use in our research?
Sfi used to cut Duckweed DNA and
plasmid for joining
Cloning W.a. cDNA fragments into
the pDNR-Lib polylinker
A.f.Insert
insert
Ava1-cuts insert out of
plasmid
AvaI CPyCGPuG
Py stands for pyrimidine- T or C
Pu stands for purine - A or G
CTCGAG
CTCGGG
CCCGAG
CCCGGG
Information on
Restriction Enzymes
Serve as landmarks in
plasmid to help find insert
SMA I
ECORI
XBA I
XHO I
HIND III
CCCGGG
GAATTC
TCTAGA
CTCGAG
AAGCTT
What will be our first step?
We will start with a DIGEST
Cutting our insert out of the
pDNR-Lib plasmid
What is most important to
remember?
Always keep enzymes on ice
(denaturation)
Always use fresh tips
Keep record in log book
clone name
date of digest
How do we name our
clones?
13ME01.09
13=PHHS
ME=Initial of person who made clone
01=Number assigned to clone
09=Year of project
What materials should I
have for Digest?
Ice in bucket
Miniprep DNA
AVA1 enzyme
10X buffer
ddH2O
10X loading gel
Microfuge tubes
Incubator 37C
Pipetman
Pipet Tips
Sharpie
Microcentrifuge
Tube holder
Lets practice pipeting
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DIGEST PROCEDURE
Label I microfuge tube 5X
Digest mix
Label ____ tubes with clone
name and digest
DIGEST PROCEDURE
1 Reaction mix
dd H2O
7ul
10X buffer
2ul
Miniprep DNA 10ul
AVA1
1ul
5 reaction mix
dd H2O
35ul
10X buffer
10ul
Miniprep DNA *
Ava1
5ul
DIGEST PROCEDURE
Mix reaction mix by pipeting up and
down
Add 10 ul of reaction mix to each
microfuge tube labeled with a clone
name
Add 10ul of the corresponding DNA to
the corresponding labeled tube
Mix each tube by tapping or in centrifuge
at low for a few seconds
Incubate for 1hour at 37C
Add 2ul of 10X loading gel
Store in freezer -20C
Next Procedure performed
on miniprep DNA is PCR
What is PCR?
How does PCR work?
Denatures DNA
Primers anneal
Taq polymerase extends
primers
Repeat cycle 30 times
Performed in a thermocycler
SHOW ANIMATIONS
95C
50C
72C
What materials are needed
to perform PCR?
Thermocycler
ddH2O
Primer forward
Primer Reverse
Miniprep DNA
Pipetman
Pipet tips
Vortex
Microfuge tubes
Ice with bucket
Rack for
microfuge tube
Rack for PCR Tube
PCR tube with
bead
Bead contains
(taq polymerase,
buffer, and
nucleotides)
PCR PROCEDURE
Label four tubes 50 fold dilution
and clone name
Label PCR tubes with clone
name
Label 1 tube 5RX mix PCR
DILUTE DNA FOR PCR
To each tube labeled 50 fold
dilution add:
98ul of ddH2O
2ul corresponding miniprep
DNA
Mix by vortexing
WHY DILUTE
PCR PROCEEDURE
1 REACTION MIX
ddH2O
18ul
Forward Primer
2.5ul
Reverse Primer
2.5ul
DNA (diluted)
2ul
5 REACTION MIX
ddH2O
90ul
Forward Primer
12.5ul
Reverse Primer
12.5ul
DNA
*
PCR PROCEDURE
Mix reaction mix by vortexing
Add 23ul of reaction mix to each
labeled PCR tube
Add 2ul of diluted DNA to
appropriate PCR tube
Mix by gentle tapping
Place in thermocycler
Record location in thermocycler