VIRAL DNA PURIFICATION
“Trino’s Lab”
Page 209-231
FAMILY: Geminiviridae
Genus: Begomovirus
Species: Tomato golden mosaic
virus (TGMV)
This Lab
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Three weeks
Report 43 points
Pre Lab 9 points
Total 52
Properties of DNA
• Large polymers made up of nitrogenous
bases and 5 carbon sugars linked by
phosphodiester bonds.
• Nucleic acids because of phosphate
groups with pKa of 1-2. Produces a strong
acid with net – charge at neutral pH
Amax Protein
Average Amax is at A260
Properties of DNA
•BORING
• But……………………..
Relatively easy to Isolate
• Obtained in very high molecular weight
form.
– DNA Molecule is designed to retain integrity
• mRNA by contrast intended to be degraded
• Protein subject to attack by enzymes and may be
pH ionic strength and temperature sensitive
– Proteins have personality
• Different protocols are designed for
different sources of DNA not differences in
the molecule.
GEMINIVIRAL DNA
PURIFICATION
• GEMINIVIRUSES:
– Plant DNA viruses
– ssDNA (most plant viruses are RNA)
– Replication in nuclei
– Whitefly transmitted
– Problem worldwide
GEMINIVIRAL DNA
PURIFICATION
• Geminiviruses are
named by its shape
under electron
microscope
GEMINIVIRAL DNA
PURIFICATION
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Four genus:
Mastrevirus
Curtovirus
Topocuvirus
Begomovirus:
Begomovirus are
whitefly transmitted
GEMINIVIRAL DNA
PURIFICATION
• Begomovirus infect dicot plant species, such
as tomato, potato, tobacco, pepper, cassava,
cotton and many others.
Leaf Morphology
Uninfected
Infected
GEMINIVIRUS DNA
PURIFICATION
• Begomovirus contain a circular ssDNA
genome that can be mono or bipartite, of
about 2.5 kb each.
• TGMV (Tomato Golden Mosaic Virus) is a
bipartite geminivirus
• They code for only 6 genes.
• Replicated by plant machinery in nuclei
GEMINIVIRUS DNA
PURIFICATION
• Big differences
among animal and
plant tissue can make
difficult to extract
nucleic acids.
– Cell wall
– Polysacharides
– Nucleases
• DNA POPULATIONS:
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Chromosomal DNA
Mitochondrial DNA
Chloroplast DNA
Viral DNA
Schedule
• Day one: First two pages of protocol( We will do the dry
blot which will sit overnight)
Day two: U V Fixation Prepare the probe that will be
used in day three. You will do Hybridization ( The
hybridization will run overnight)
Day three: Complete Hybridization, detection and photo
record.
• Read introduction and theory Page 211-221
• Read protocol summary Page 222-223
Day one: Extraction of DNA from
infected leaf (Page 224)
• One leaf infected; one uninfected
• Grind by rotation while pressing down in 250
microliters of extraction buffer
– Extraction Buffer (3NO)
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7 M Urea
0.35 M NaCl
0.05 M Tris HCl pH 8
0.02 M EDTA
1% Sarkosyl (Fisher #BP234-500)
• After homogenized add buffer to total volume of
750 microliters
• Mix well and wait 30 min
Protocol
• Very typical this basic approach can be
used to purify smaller DNAs from virtually
any source (Hirt extraction)and large
RNAs from bacteria
• Like the LDH purification begin by grinding
tissue.
• Conduct a chloroform/phenol extraction
– Save aqueous phase
• PPT the DNA with alcohol
GEMINIVIRAL DNA PURIFICATION
(Bench protocol Page 412)
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Homogenize sample (with pestle) in buffer (step 1-4)
Incubate (10 min at 65C Step 5)
Centrifuge 2min (6)Save supernatent
Mix supernatant with phenol:chloroform (7)
– Note: Protocol says add equal amount. A little more
is better
Centrifuge 2 min (8)
Separate aqueous phase (9)
Isopropanol and salt precipitation (10)
Ethanol wash (step 12)
• Dry and resuspend in dd water (Step 14)
Gel separation of virus DNA
• GEL PREPARATION AND RUNNING:
– Agarose melting and mix with Ethidium
Bromide (MUTAGENIC, WARNING !!!)
– Pouring, wait until is solid.
– Mix your DNA from purification with loading
buffer and add to gel.
– Set 120 Volts constant, run for 30 minutes
– Take photographic register of the gel
Denaturing and preparation of DNA
for transfer to membrane
• Denature DNA in gel (Step 1-5)
• Neutralize (step 6-7)
• Set up transfer to nylon membrane
Transfer of DNA from gel to
nylon membrane
• SOUTHERN TRANSFER:
– Cut the Nylon membrane to the same size of
the gel (nylon is positively charged)
– Cut filter paper and absorbent paper enough
to make a 10cm pile (about 2-3 inches).
Transfer of DNA from Gel to
Membrane
1" stack of cut
paper towel pieces
Whatman 3mm
Nylon membrane
Agarose gel with
resolved DNA fragments
Whatman 3 mm
Uprighted
pipet tip box
Allow Transfer to Occur
Overnight
End of Day One
Protein Purification Report
• Will Be Due
• Nov9/10(next week)
Gemini Virus DNA Report
Will be due the Day that QPCR
experiment is conducted
Oct 23/24
Day Two
Membrane fixation, Probe
preparation, Hybridization
Preparation of probe and
hybridization
• UV Fixation (page 413 step 10)
– Once the gel has been transferred (about 8-16
hours), take the Nylon membrane and expose it to UV
light, using a crosslinking cabnet.
– Set the crosslinker to optimal.
– Instructor will do this step
• TA will prepare probe
• Week two: Detection, Probe with a SS DNA
complementary to Gemini virus DNA (page 413
step 1-3)
Probe preparation:Protocol
AseI
Restriction
endonuclease
treatment
NcoI
Gemini virus
sequence-containing
plasmid
AseI
AseI
NcoI
NcoI
+
Replication
using digoxigenin
-labeled UTP
DG-labeled
probe DNA
•Produced an 800 BP long probe labeled by incorporation of
NTPs labeled with digoxigenin.
DU DU
DU
Reminder:
PCR
Digoxigenin Reaction
You will use probe to detect virus
DNA in your blot (page 196 steps
1-9)
Viral DNA bound to
DU-labeled probe DNA
DU
DU
This is a Southern hybridization
DNA to DNA. Probe binds by base pairing
with Denatured DNA on the nylon filter
DU
DU
An antibody can detect the DU labled probe
Hybridization
• Requires extended time at moderate
temperature
– Selects for accurate hybrids
• Leave overnight Page 414 step 5
• Ta will complete steps 6,7,8&9
• End of Day two
Day Three
Identification of Virus DNA in mixture of
host cell DNA by antibody binding to
DU labled Gemini specific probe
Day three
• Detection of virus DNA
• Use DU specific antibody conjugate
– Page 196 steps 1-8
• Record by digital photography step 9 &10
Immuno-detection of virus specific
probe
Substrate
Immunoconjugate
E
Viral DNA bound to
DU-labeled probe DNA
Product
(colored ppt.)
DU
DU
DU
DU
From Last Year
M
I
M
I
M=Not Infected
I = Infected
DS DNA
SS DNA
Week 3
• TA will complete steps 1&2 page 414
• Add 5ml of blocking buffer containing 1 microliter of
antibody
– Incubate 30 min
– Pour into sink
• Wash twice with 1X washing solution
• Add 3 ml detection buffer
– Rock for 4 minutes
• Add 5ml of substrate
• Wait 30min. Check for detection
• Rx is complete in 5 hours
Next Week
• QPCR
• Lab D3 Page 253
• Read introduction and Theory
– Page 258-264
• Protocol page 266