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

Average Amax is at A260

Amax Protein

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 cutpaper towel pieces

Whatman 3mm

Nylon membrane

Agarose gel withresolved DNA fragments

Whatman 3 mm

Uprightedpipet tip box

Allow Transfer to Occur Overnight

End of Day One

Protein Purification Report• Will Be Due• April 13/14• (second week of the virus lab)

Gemini Virus DNA Report

Will be due day of the second examApril 29(001)/30 (002) at 1:00pm Dabney 308

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

DUDU DU

+

Restriction endonuclease treatment

Replication using digoxigenin -labeled UTP

Gemini virus sequence-containing plasmid

AseI

NcoI NcoI

NcoI

AseI

AseI

DG-labeled probe DNA

•Produced an 800 BP long probe labeled by incorporation of NTPs labeled with digoxigenin.

Reminder: PCR

Pcr.exe

Digoxigenin Reaction

You will use probe to detect virus DNA in your blot (page 196 steps

1-9)

DU

DUDU

DU

Viral DNA bound toDU-labeled probe DNA

This is a Southern hybridization DNA to DNA. Probe binds by base pairing with Denatured DNA on the nylon filter

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

DU

DU

DU

DU

E

Substrate

Product(colored ppt.)

Viral DNA bound toDU-labeled probe DNA

Immunoconjugate

From Last Year

M I M I

M=Not InfectedI = Infected

DS DNASS 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 45 points

• Pre Lab 9 points

Next Week

• QPCR

• Lab D3 Page 253

• Read introduction and Theory– Page 258-264

• Protocol page 266