This video demonstrates a procedure for separating mixed viral assemblages based on their nucleic acid composition. This enables the study of the taxonomic diversity and functional potential of complex viral assemblages. Here hydroxyapatite, a crystalline form of calcium phosphate is employed in the separation of nucleic acids.
First, a Hydroxyapatite column is packed and equilibrated purified viral nucleic acids are then applied to the column, the charge interaction between the positively charged calcium ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes binds the nucleic acids to the column Nick. Next single stranded, D-N-A-R-N-A and double stranded DNA are sequentially alluded with different concentrations of phosphate buffer. The resulting nucleic acids are then desalted and concentrated in preparation for visualization, quantification and identification by DNA sequencing agros gel electrophoresis confirms that the nucleic acid species can be separated with less than 9%carryover.
Thus, this method enables the evaluation of the diversity and functional capacity of viral communities. Hello, my name is Doug Fro, and I'm a research associate in the lab of Dr.Shannon Williamson at the j Craig Venture Institute. Today I will be showing you a procedure to separate single stranded DNA double stranded DNA and RNA from a environmental viral assemblage.
This method has advantages over existing methods such as the treatment with the nucleus, which targets either DNA or RNA. Hydroxyapatite chromatography has the ability to capture the entire viral community. This method allows us to answer several questions in the field of viral ecology.
For example, what is the total gene complement or the diversity of the viral community? We first thought of this method when we wanted to study the entire Rio Plankton assemblages from the Chesapeake Bay without sacrificing a subset of the the genetic signature. The solutions needed for this experiment should be prepared ahead of time according to the instructions in the accompanying written protocol, and include 0.12 molar phosphate buffer, 0.20 molar phosphate buffer, 0.40 molar phosphate buffer, and one molar phosphate solution.
All at pH 6.8 and hydrated hydroxyapatite on the day of the experiment, equilibrate all phosphate buffers and hydrated hydroxyapatite by placing them in a 60 degree Celsius water bath. Begin this procedure by preparing the econo column that will be used for the separation of viral nucleic acids. Attach a stopcock to the end of the econo column and ensure that it is closed.
Using a pipetter add deionized water to the column, then place the cap on the top of the column and repeatedly invert the column to rinse it. Pour off the water and repeat this wash several times. Next, remove the stop cock from the econ column and autoclave it to sterilize it.
The stop cock is not autoclave since it will melt. Rinse the stop cock with the ionized water, then again with 70%ethanol and allow it to air dry after autoclaving. Replace the stop cock and close it.
Next, working in a fume hood, add one milliliter of sigma coat to the column and coat the column glass by inverting the column with a rolling motion as shown here. Coating with a sigma coat will minimize sticking of the nucleic acids to the glass surface. During separation, one salt glass surfaces have been coated.
Open the stop cock and decant into a conical tube. Next, to connect the column to a circulating water bath, use two clamps to attach the column to a ring stand. Attach silicon tubing from the circulating water bath, so the flow of the water from the water bath is from bottom to top.
Set the temperature to 60 degrees Celsius and start the circulation. While in practice, the column and the tubing would be wrapped in an insulating agent, such as aluminum foil or foam pipe insulation. To minimize heat loss, this step is not shown here.
In order to better visualize the procedure, close the stop cock. Using a pipetter, add 10 milliliters of sterile RNAs free water. Open the stop cock and allow the water to drain.
Then wash again following the second wash. Rinse twice with 10 milliliters of sterile RNAs free 0.12 molar phosphate buffer and decant between washes. The column is now ready for hydroxyapatite chromatography.
Ensure that the econ column stop cock is closed. Then using a sterile two milliliter serological pipette slowly add two milliliters of prepared resuspended rehydrated hydroxy appetite to the bottom of the column. Replace the column cap.
Allow the hydroxy appetite to settle under the force of gravity for 30 minutes. This method requires attention to detail. The hydroxy appetite column processes at a rate of 0.25 to 0.5 mils per minute.
So it's very important to keep a close eye on the column so it does not dry out. After 30 minutes has passed, open the stop cock to drain the buffer into a waste container. Close the stop cock and discard the flow through.
Combine nucleic acids with 0.12 molar phosphate buffer in a final volume of 500 microliters and incubate it 60 degrees Celsius for 10 minutes. This will equilibrate the nucleic acids to the temperature of the column and will enhance the separation of the different nucleic acid species by removing any secondary structures that may be present. Using a two milliliter serological pipette, quickly apply the nucleic acid solution to the hydroxy appetite, making sure not to disturb the column.
Once the entire 500 microliters flows onto the column, a small amount of 0.12 molar phosphate buffer can be added to the top of the column to prevent it from drying out. Allow the nucleic acids to bind to the hydroxy appetite for 30 minutes. Following the binding incubation, place a 15 milliliter conical tube under the column.
Then open the stop cock and collect the initial 500 microliter sample, which contains single stranded DNA. Once 500 microliters has been collected, close the stop cock. Next, add six milliliters of 0.12 molar phosphate buffer to the hydroxy appetite, making sure not to disturb the column.
Then open the stop cock and continue to collect the flow through containing singlet stranded DNA in the same 15 milliliter tube from the previous step. After approximately six milliliters has been collected, close the stopcock. Add one volume of a phenol chloroform isoamyl alcohol solution to the tube mixed vigorously by vortexing, then centrifuge at 3, 500 times G for 15 minutes.
Following the centrifugation, the tube will contain two layers. Transfer the top acquiesce layer, which contains the single strand of DNA to a new 15 milliliter tube, and store the sample at room temperature to elute and purify RNA From the column, add six milliliters of 0.20 molar phosphate buffer and collect the flow throughs before using a new 15 milliliter tube. Then perform a phenol chloroform extraction.
As for the single stranded DNA sample, repeat this process using six milliliters of 0.40 molar phosphate buffer to elute and purify double stranded DNA from the column. Finally, to strip the column of any residual doublet stranded DNA. Repeat this process one more time.
This time using six milliliters of 1.00 molar phosphate buffer to desalt the nucleic acid samples. Transfer four to five milliliters of each sample to anon Ultra four centrifugal filter device outfitted with a 30, 000 molecular weight cutoff ultra cell membrane concentrate sample to less than 500 microliters by spinning at 6, 000 GS at 30 degrees Celsius for about five minutes in a fixed angle rotor. After discarding the flow through, add the remainder of the samples to the filter.
Bring the volume up to four to five milliliters with RNAs free one XTE buffer. Then spin again until a concentrated volume is achieved. About five minutes following the spin, add four to five milliliters of RNAs free one XTE buffer to the filter and concentrate.
Again, repeat this wash and additional five times to completely desalt nucleic acid samples. Once the samples have been completely concentrated and desalted transfer the remaining nucleic acid samples to a sterile 1.7 milliliter micro centrifuge tube. Precipitate the nucleic acids by adding one 10th of the volume sodium acetate, two volumes of 100%ethanol and one microliter of glyco blue mixed well by vortexing centrifuge.
A 28, 000 GS four degrees Celsius. 60 minutes to Kent. Making sure not to disturb the pellet.
Add 300 microliters of 70%ethanol spin at 28, 000 GS room temperature for 10 minutes to can't dry and resuspend each fraction in an appropriate volume of RNAs. Free TE buffer equal concentrations of the viral nucleic acids M 13 MP 18, single stranded DNA MS two single stranded, RNA FI six double stranded, RNA and lambda double stranded DNA were combined, then separated using hydroxyapatite chromatography as shown in this video. Equal concentrations of M 13 MP 18, single stranded DNA MS two, single stranded, RNA five six double stranded, RNA and Lambda double stranded DNA were combined as shown in lane six and applied to a hydroxyapatite column as can be seen here.
Single stranded D-N-A-R-N-A and double stranded DNA were independently alluded from the Hydroxyapatite column using 0.12 molar 0.18 molar 0.40 molar and 1.00 molar phosphate buffer. A one KB ladder was used to confirm genome sizes. These results indicate that this method can be used to successfully separate single stranded DNA double stranded DNA and RNA with less than 9%carryover from one fraction to the next.
This method was also used to separate nucleic acids isolated from a viral community within the Chesapeake Bay, as shown here. Single stranded D-N-A-R-N-A and double stranded DNA were independently alluded using phosphate buffer concentrations of 0.12 molar 0.20 molar and 0.40 molar, or 1.00 molar respectively high mass DNA ladder in lane one and one KB ladder in lane two were used to visually confirm approximate molecular weight and mass of nucleic acids as can be seen here, double stranded DNA was the dominant nucleic acid type extracted from the Chesapeake Bay Verio plankton. After watching this video, you should have a good understanding of how to separate nucleic acids from a mixed viral assemblage using hydroxyapatite chromatography.
This includes the preparation of the Hydroxyapatite column and nucleic acids, the separation and illusion of nucleic acids using various concentrations of phosphate buffer and desalting, and concentrating the nucleic acids after separation.
