This method can help answer key questions about environmental virology, such as the emerging of new diseases. The main advantage of this method is purifying variants away from cellular components and using those variants to extract DNA for sequencing. I first came to this idea when we found out that the plants in our greenhouses infected with one or more unknown Badnaviruses.
Individuals new to this method will struggle for three reasons. First, homogenization is a critical step to ensure quality yields. Second, differential centrifugation can produce multiple pellets and you need to know how to identify and separate those pellets.
Third, bio-analyzer can also be difficult to interpret. A laboratory coat and gloves should be worn for all steps of this procedure. Start by cutting 80 to 100 grams of leaves from diseased plants previously frozen at minus 20 degrees Celsius.
Then grind the leaves in a Waring blender using 200 milliliters of grinding buffer and 0.5%sodium sulfite. Transfer the homogenate to a one liter beaker. While still wearing appropriate personal protective equipment, add 18 grams of urea and 25 milliliters of 10%non-ionic detergent to the homogenate and cover the beaker with foil.
Stir with a magnetic stirrer overnight. On the following day, transfer the homogenate to 250 milliliter centrifuge rotor bottles and centrifuge in a fixed angle rotor at 4000 times g at four degrees Celsius for 10 minutes. Recover the supernatant and filter through four layers of cheese cloth.
Measure the volume of the recovered liquid homogenate and then divide the homogenate among 38.5 milliliter polypropylene centrifuge tubes. Centrifuge the homogenate at 40, 000 times g at four degrees Celsius for 2.5 hours. When the centrifuge is done, check for the presence of a green pellet at the bottom of the tube and a white pellet along the length of the tube.
Pour off the supernatant, retaining both pellets, and place the samples on ice. Separating the white and the green pellet is one of the most challenging steps in the procedure. It requires patience and skill.
Working in a chemical hood, use a rubber policeman to separate the pellets. Scrape and transfer the pellets to different rotor bottles. Place the rotor bottles containing the white pellets on ice.
Add one milliliter of water to each bottle and pipette up and down. Every five minutes, over the course of one to two hours, pipette the mixture up and down to re-suspend the pellet. After the white pellets have been re-suspended, keep the suspensions overnight at four degrees Celsius.
On the following day, centrifuge the suspension at 6000 times g at four degrees Celsius for 10 minutes to remove the remaining debris. Transfer the concentrated suspensions to new tubes and centrifuge at 136, 000 times g at four degrees Celsius for two hours to pellet virions. Remove the supernatant and re-suspend the pellet in one milliliter of buffer.
Begin this procedure by disrupting the virions. Add four microliters of a two microgram per microliter proteinase K solution to the pellet suspensions and incubate at 37 degrees Celsius for 15 minutes. Add one volume of phenol chloroform isoamyl alcohol to each sample and shake by hand for 20 seconds.
Centrifuge at room temperature at 16, 000 times g for five minutes. Remove the upper aqueous phase and transfer to a new tube. Repeat this extraction two or more times.
Concentrate the DNA using ethanol precipitation. Use 0.3 molar of sodium acetate pH 5.2 and 2.5 volumes of 95%ethanol. Place the samples at minus 20 degrees Celsius for 30 to 60 minutes.
Then, centrifuge at 13, 000 times g for 10 to 20 minutes to pellet the DNA. Working at a laboratory bench, re-suspend the DNA pellet in one milliliter of 0.1 millimolar TE buffer. Filter the suspension through a commercial gel filtration column by centrifugation.
Analyze the samples by 1%agarose gel electrophoresis using ethidium bromide staining to view the quality preparations and assess the quality of the DNA as outlined in the text. When examined by transmission electron microscopy, virus particles were present in the white pellet recovered by crude fractionation of infected canna leaves. Agarose gel electrophoresis of DNA recovered from the green and white pellets identified two high molecular weight DNA bands in the white fraction indicated by the red and yellow dots.
The integrity of RNA isolated from infected leaves was also by verified agarose gel electrophoresis. This chart shows the abundance and taxonomic distribution of contigs assembled from the crude virus preparation. And this chart depicts the proportions of virus contigs associated with the Caulimoviridae family, Badnavirus genus and contigs associated with three closely related species.
These next two charts show the abundance of contigs derived from RNA sequencing based on their taxonomic distribution. The chart on the right depicts the abundance of contigs within the population of virus contigs associated with the Caulimoviridae family Badnavirus genus and three closely related species. Comparing the virus genome length contigs produced by DNA and RNA sequencing as a mutual scaffold confirm the presence of two full length virus genomes.
Finally, RT PCR analysis using RNA isolated from virus-infected leaves detected both virus genomes. Once mastered, this technique can take two days, whereas a beginner may take three days. While carrying out this procedure for the first time, it's important to keep records of the volume of homogenate in each of the supernatants so at the end of the procedure you can compare the final yield of DNA with the initial starting material.
After its discovery, this method has paved the way in the field of plant virology for researchers to identify new DNA viruses in the environment. After watching this video, you should have a good understanding of how to homogenize plant material, concentrate variants, and obtain a good yield of DNA that can be used for next generation sequencing. Don't forget that working with the urea and phenol chloroform is extremely hazardous and a precaution such as wearing a goggle, a blouse and lab coat, and a mask should be taken always when you are working with these chemicals.
For safe ultra centrifugation, it is important to properly balance the tubes, so you need to weigh them and place them opposing to each other in the rocker as well as the weight should be between 0.01 of each other.
