This method can help answer key questions in the HIV field such as determining the genetic composition of HIV proviruses and different cell types. Especially identifying those that are genetically intact and potentially replication competent. The main advantages of this technique is that it allows us to amplify near full length HIV proviruses from single HIV infected cells and then sequence these by next generation sequencing.
To begin, prepare all buffers as outlined in the text protocol. Obtain a cell pellet and add one hundred microliters of lysis buffer per million cells. Mix by pipetting up and down.
Lyse the cells by incubating in a thermal cycler at 55 degrees celsius for one hour and then add 85 degrees celsius for 15 minutes. To begin amplifying single HIV-1 DNA proviruses mix the reagents for the first round PCR as listed in table one of the text protocol. Add 38 microliters to 85 wells in a 96 well PCR plate and label this plate as PCR 1.
After this, move the PCR 1 plate to a clear area designated for the addition of genomic DNA. Using tris hydrochloride serially dilute the genomic DNA from a ratio of one to three to a ratio of one to 81 making sure to prepare 45 microliters of each dilution. Add two microliters of diluted genomic DNA to each sample well and two microliters of tris hydrochloride to each negative control well.
Seal the entire plate except for the positive control well. Next, move to an area designated for the addition of the positive control. Add two microliters of the positive control to the corresponding well and then seal the plate completely.
Use a PCR plate spinner to briefly spin the PRC 1 plate and pull down any residual contents from the sides of the wells. Run the PCR 1 plate in the thermal cycler as outlined in the text protocol. Next, mix the reagents for the second round of PCR as listed in table one.
Add 28 microliters of this PCR 2 mix to 85 wells of a new 96 well PCR plate. Label this plate as PCR 2. Using a plate spinner, briefly spin the PCR 1 plate to pull down any residual contents from the sides of the wells.
Add 80 microliters of tris hydrochloride to each well of this plate. After this, use a mutli-channel pipette to transfer two microliters from each well of the PCR 1 plate to the corresponding wells in the PCR 2 plate. Seal the PCR 2 plate with clear adhesive wrap.
Briefly spin the PCR 2 plate in the plate spinner. Meanwhile, seal the PCR 1 plate with a heat sealing film for longterm storage at minus 20 degrees celsius. Run the PCR 2 plate in a thermal cycler as outlined in the text protocol.
Then briefly spin the PCR 2 plate to pull down any residual contents from the sides of the wells. Using a multi-channel pipette, add 60 microliters of tris hydrochloride to each well. Next, run 15 microliters of each well on two 48 well precast one percent agarose gels that contain ethidium bromide.
Identify the wells containing the amplified product and their approximate sizes and save the gel image. After this, determine the dilution at which no more than 30 percent of wells are positive for amplified product. First, use a plate spinner to briefly spin the PCR 2 plate and pull down any residual contents from the sides of the wells.
Transfer 40 microliters from the wells containing amplified product to the corresponding wells of a new 96 well midi plate. Next, set out a magnetic feed based purification kit making sure to bring the magnetic beads to room temperature and prepare a fresh solution of 80 percent ethanol. Once the beads reach room temperature vortex them to ensure that they are thoroughly resuspended.
Using a multi-channel pipette, add 40 microliters of beads to the 40 microliters of amplified product in each well of the midi plate. Mix by gently pipetting up and down 10 times. Incubate at room temperature for five minutes.
Then, transfer the plate to a magnetic stand and let it rest for two minutes. After this, remove and discard the supernatant. With the plate still on the stand, wash the beads by adding two hundred microliters of the 80 percent ethanol solution to each well.
Incubate at room temperature for 30 seconds and then remove and discard the supernatant. Repeat this wash process once from adding the ethanol to discarding the supernatant. Using a multi-channel pipette remove any excess ethanol.
Let the beads air dry for 15 minutes. After this, remove the plate from the stand. Add 30 microliters of elution buffer to each well and mix by gently pipetting up and down 10 times.
Incubate at room temperature for two minutes. Place the plate back on the magnetic stand and let it rest for two minutes. Using a multi-channel pipette, transfer 25 microliters of the supernatant to the corresponding wells of a new 96 well PCR plate.
Next, use a spectra photometer to determine and record the approximate concentration of each amplified DNA product. Set out a double stranded DNA quantification kit and dilute the buffer to one times in sterile DNAs-free water. Add 99 microliters of the buffer to an appropriate number of empty wells in a flat bottom tissue culture plate.
Then, add one hundred microliters of buffer to three blank wells. For each amplified product to be measured add one microliter of purified DNA in triplicate to each well containing buffer. Dilute lambda double stranded DNA 10 fold from two nanograms per microliter to point zero zero nanograms per microliter to prepare the standards.
Add one hundred microliters of each double stranded DNA standard to three wells. Dilute the florescence dye one to two hundred with buffer. Quickly add one hundred microliters to each well that contains a sample, blank or standard and mix by pipetting up and down.
After this, cover the plate with foil to avoid contact with light. Using a microplate reader, record the florescence emission. Use the recorded measurements to determine the concentration of double stranded DNA in each sample.
Then dilute each purified product with water to a concentration of zero point two nanograms per microliter. Following nested PCR, the amplified products are run on a one percent agarose gel. The initial quality of the PCR can be determined by inspecting the controls.
As negative controls containing amplified product indicate contamination and the positive controls without amplified product indicate insufficient amplification. Only wells run at end point dilution that contain single amplified proviruses are considered for sequencing. While wells containing multiple amplified proviruses of different lengths can be visualized at this stage, they should not be selected for sequencing.
For the de novo assembly, the quality of the assembled contigs can be assessed for sufficient depth and evenness of coverage. Mixed populations can also be identified at this stage by the presence of uneven coverage. Visual representation of the sequences isolated from one participant reveals that 97 percent of their sequences are defective.
With intact sequences found in effector and transitional memory CD4 positive T cells. While attempting this procedure it's important to remember that only amplified products obtained at the limiting dilution, that is where no more than 30 percent of the wells are positive, contain a single provirus. Following this procedure, amplified products can be sequenced to determine the genetic composition of HIV proviruses.
After its development, this technique paved the way for researchers in the field of HIV to explore the distribution of genetically intact HIV proviruses and different cell types in patients on therapy to determine where genetically intact and potentially replication competent proviruses hide. Don't forget that working with HIV infected cells can be hazardous and precautions such as wearing appropriate personal protective equipment and working a certified laboratory should always be taken while performing this procedure.
