INTRODUCTION TO THE WAKSMAN RESEARCH PROJECT

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INTRODUCTION TO THE WAKSMAN RESEARCH PROJECT

• DNA Sequence Analysis of the Duckweed Wolffia
  arrhiza

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PROBLEM

• What genes are present in Wolffia arrhiza?
• What are the functions of these genes?

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WHY DO WE WANT TO IDENTIFY GENES AND THEIR
FUNCTIONS?
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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

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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

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Why Wolffia arrhiza?
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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

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Duckweed may serve as a model organism
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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

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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

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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

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In order to study DNA, it must be amplified and
eventually purified and stored
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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.
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Because genes are composed of As, Cs, Gs, and
Ts, 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?

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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?

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VECTORS ALLOW US TO ACCOMPLISH ALL THREE TASKS
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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

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Amplification can be accomplished thru cloning
with a vector
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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)

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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

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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

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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

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What is a Plasmid?

• Replicated by the hosts 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

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What is a plasmid?
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Two Types of plasmids
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What type of plasmid are we using?
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What are important properties of pDNR-Lib?
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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.

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Plasmid Characteristics

• Ori
• Selectable Marker
• 3.6 Kb
• Color screen (not in this plasmid)
• MCS
• Circular DNA

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Color Screen-not found in pDNR-LIB but important
in many other types of plasmids
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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

• Transformation-introduction of foreign plasmid
  into a bacteria

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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

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Where do restriction enzymes come from?
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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
• 464,096 bp

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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

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What happens if we cut Duckweed DNA and our
plasmid with EcoR1?

• Example on board

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Why do we add ligase?

• Link together nucleotides
• Phosphodiester bonds
• Dehydration synthesis

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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

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What restriction enzymes do we use in our
research?
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Sfi used to cut Duckweed DNA and plasmid for
joining
Cloning W.a. cDNA fragments into the pDNR-Lib
polylinker
A.f. insert
Insert
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Ava1-cuts insert out of plasmid

• AvaI CPyCGPuG CTCGAG
• Py stands for pyrimidine- T or C CTCGGG
• Pu stands for purine - A or G CCCGAG
• CCCGGG

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Information on Restriction Enzymes
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Serve as landmarks in plasmid to help find insert

• SMA I CCCGGG
• ECORI GAATTC
• XBA I TCTAGA
• XHO I CTCGAG
• HIND III AAGCTT

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What will be our first step?

• We will start with a DIGEST
• Cutting our insert out of the pDNR-Lib plasmid

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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

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How do we name our clones?

• 13ME01.09
• 13PHHS
• MEInitial of person who made clone
• 01Number assigned to clone
• 09Year of project

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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

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Lets practice pipeting
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DIGEST PROCEDURE

• Label I microfuge tube 5X Digest mix
• Label ____ tubes with clone name and digest

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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

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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

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Next Procedure performed on miniprep DNA is PCR
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What is PCR?
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How does PCR work?

• 95C Denatures DNA
• 50C Primers anneal
• 72C Taq polymerase extends primers
• Repeat cycle 30 times
• Performed in a thermocycler
• SHOW ANIMATIONS

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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)

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PCR PROCEDURE

• Label four tubes 50 fold dilution and clone name
• Label PCR tubes with clone name
• Label 1 tube 5RX mix PCR

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DILUTE DNA FOR PCR

• To each tube labeled 50 fold dilution add
• 98ul of ddH2O
• 2ul corresponding miniprep DNA
• Mix by vortexing
• WHY DILUTE

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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

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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