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3. genetics

VIRAL DNA PURIFICATION
“Trino’s Lab”
Page 209-231
FAMILY: Geminiviridae
Genus: Begomovirus
Species: Tomato golden mosaic
virus (TGMV)
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
•
•
•
•
•
•
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:
–
–
–
–
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
• 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)
•
•
•
•
•
•
•
•
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
Day Two
Membrane fixation, Probe
preparation, Hybridization
Gemini Virus DNA Gel
M
I
I
U
U
M
M=marker, I= infected, U= uninfected, Arrow indicates virus RNA
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
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
This Lab
• Three weeks
• Report 40 points
• Pre Lab 9 points
Next Week
• QPCR
• Lab D3 Page 253
• Read introduction and Theory
– Page 258-264
• Protocol page 266