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
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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