This new method for analyzing reverse transcription can tell us new things about the field of retro virology. For example, we can learn about the details of how cellular restrictions factors work or how antiretroviral drugs inhibit HIV1 DNA synthesis. The main advantage of this technique is that not only provides information about the sequence of Nascent Reverse Tran scrips, but it allows mapping of their precise 3-prime DNA termini at single nuclear tight resolution.
The enrichment of HIV1 complimentary DNAs from wholesale DNA of HIV1 infected cells, is done by hybrid capture and should be carried out in a PCR workstation. Prepare a master mix of magnetic streptavidin beads by pipe heading one hundred microliters of beads per sample into a single micro centrifuge tube place the tube on a magnet suitable for micro centrifuge tubes. After the beads have settled toward the magnet side of the tube, remove the storage buffer, remove the tube from the magnet, and re suspend the beads in five hundred microliters of bindent wash or BW buffer.
Place the tube back on the magnet, wait for the beads to migrate to the magnet, and remove the supernatant. Remove the tube from the magnet, add five hundred microliters of casein solution, re suspend the beads, and incubate at room temperature for ten minutes. After ten minutes, wash the beads with BW buffer.
Place the tube back on the magnet, remove the supernatant and re suspend the beads in five hundred microliters of BW buffer. Add fifty picomoles of each captured biotinylated oligonucleotide per sample. Incubate at room temperature while rocking in an end over end mixer for thirty minutes.
Next, return the tube to the magnet, remove the supernatant, remove the tube from the magnet, add five hundred microliters of 1x ten buffer, and re suspend the beads. After the second wash re suspend the beads with the immobilized oligonucleotides in ten microliters of 1x ten buffer per sample. For each sample, label one microcetrofuge tube and add ten microliters of bead suspension, one hundred and seventy microliters of DNA, and ninety microliters of 3x ten buffer.
Incubate in a dry heat block at ninety two degrees celsius for two minutes to de nature the DNA. Move the tubes to a different dry heat block, which is set to fifty two degrees Celsius, and incubate for one hour. Every ten minutes during this incubation, invert the tubes to mix.
When the one hour incubation is done, wash the beads once with five hundred microliters of 1x ten buffer, and re suspend in thirty five microliters of nuclease free water. Ensure the beads settle well by leaving the tubes on the magnet for about three minutes before removing any liquid. To elute, incubate the tubes at ninety two degrees Celsius in a dry heat block for two minutes.
Then, quickly move the tubes onto the magnet, one tube at a time. Once the beads are bound to the side of the tube, transfer the supernatant containing the HIV1 DNA to a fresh tube. To begin this procedure, re suspend the lyophilized adaptor at one hundred micromolar in nuclease free water and vortex the tube.
Per sample, plus one control sample, combine zero point four five microliters of 10x T4 DNA ligase buffer, four microliters of adapter, and point zero five microliters of nuclease free water. Heat to ninety two degrees Celsius for two minutes, and then let it cool down slowly to sixteen degrees Celsius in a PCR machine. This will allow the adaptor to form a hair pin structure.
The key step in this procedure is the efficient and unbiased ligation of adapted molecules to open 3-prime DNA termini. Purified nascent cDNA molecules of varying length are ligated to a hairpin single stranded DNA adaptor using T4 DNA ligase. The adaptor carries a random six nucleotide barcode sequence, which allows for paring to facilitate ligation, and simultaneously serves as an identifier for unique reads.
The 3-prime termini adaptor carries a spacer to prevent self ligation. To set up sixty microliter final volume ligation reactions, add the following to each PCR tube:six microliters of 10x T4 DNA ligase buffer, twenty four microliters of forty percent PEG, six microliters of five molar Betaine, four point five microliters of adaptor, one point two microliters of T4 DNA ligase, and eighteen point thee microliters of DNA. After mixing the reactions well, incubate in a PCR machine at sixteen degrees Celsius overnight.
On the day after the adaptor ligation, at thirty microliters of formamide containing DNA gel loading buffer to each ligation reaction and mix well by pipe heading. Heat in a PCR machine at ninety four degrees Celsius for two minutes then immediately put on ice. The next step is to prepare for denaturing gel electrophoresis.
Place a pre cast six percent TBE denaturing urea polyacrylamide gel in an appropriate gel tank and add 1x TBE running buffer. Pre run the gel for twenty minutes. Next, use a syringe and a twenty one gauge needle to wash out the gel pockets with running buffer.
Then load thirty microliters per well of the same sample into three wells. It is advisable to only run one sample per gel to avoid cross contamination. Run for twenty minutes.
While the gel is running, prepare three zero point five milliliters microcentrifuge tubes per sample by using a twenty one gauge syringe needle to poke holes into the bottom. Insert each of the prepared tubes into a two milliliter micro centrifuge tube and label them with the sample name plus low, mid, or high. When the dark blue dye front is about half way through the gel, stop the electrophoresis and remove the gel cassette.
Pry open the cassette and cut the gel vertically with a razorblade. To generously excise the strip with three wells of loaded samples. Add the gel strip to a container with 1x TBE and five microliters of cyanine nucleic acid stain and incubate for three to five minutes.
Ensure staining is sufficient in order to easily identify the top of the free adaptor which is to be removed later. Next, clean the surface of a blue light transilluminator thoroughly with distilled de ionized water. Then take the gel piece out of the staining container and place it on the light box.
Turn on the light box and inspect the stained nucleic acids through the orange filter. A representative ligated DNA sample on a stained gel, is shown here. The adaptor typically appears overloaded and runs as a big blob with ligated HIV1 DNA running above as a streak.
The red lines indicate the sites the gel is to be cut to remove free adaptor, and divide the sample into three even pieces. Using a new razorblade, cut away the sides of the gel if there areas with no sample loaded still present. Next, cut just above the adaptor to remove the adaptor and lower gel parts.
Finally, cut away the very top of the gel including about one millimeter of the gel pockets which often have a sharp intense signal of higher molecular weight DNA. Divide the remaining gel piece containing the sample horizontally into three even pieces. Low, mid and high molecular weight areas.
Then, cut each of the three gel fragments into two by two millimeter pieces and transfer them into the prepared zero point five milliliter microcentrifuge tubes. Spin at top speed with open lids for one minute. To squeeze the gel pieces through the hole into the two milliliter tube to create a gel slush.
If any gel particles remain in the bottom of the zero point five milliliter tube, transfer them to the two milliliter tube manually using a needle. Begin the DNA extraction procedure by adding one milliliter of urea gel extraction buffer to the gel slush in each microcentrifuge tube. Rotate the tubes with an end over end mixer at room temperature for a minimum of three hours.
Prepare the filter columns. Use a clean set of tweezers to add one small round glass fiber filter to each centrifuge column with cellulose acetate membrane filters, which prevents membrane clogging. Put the filter in place with an inverted pipe head tip.
Briefly spin the two milliliter tubes with gel slush and extraction buffer in a microcentrifuge, and transfer seven hundred microliters of the supernatant to the prepared filter columns. Centrifuge the filter columns in a microcentrifuge at top speed for one minute. Then transfer the flow through to a new two milliliter microcentrifuge tube.
Reload the columns with the remaining supernatant. Try to obtain as much liquid as possible from the extraction slush and do not be concerned if gel pieces are included. Spin again.
Combine the flow through of the same extraction samples and proceed as described in the text protocol. This protocol was employed in samples from CEMS-ST cells infected with Vif-deficient HIV-1 in the absence or presence of the antiretroviral human protein A3G. A plot of the total number of unique reads obtained from each sample indicates that increasing levels of A3G reduce the total read number.
Reflecting the inhibitory effect of A3G on reverse transcriptase mediated cdna synthesis. In these next three plots, the fraction of molecules at each possible length within the first one hundred eighty two nucleotides, is shown in blue histograms. The addition of A3G cost a sharp increase of shorter truncated cdna molecules at a few very specific reproducible positions.
The dashed red lines, show the percentages of reads carrying C to T mutations at the respective position. A positive control can be produced by processing a pool of synthetic oligonucleotides of know sequence, length and concentration. All molecules should appear in close to equal abundance with only small variations.
If a library run produces a minor length bias, in sequencing, it is advisable to apply a normalization factor which is derived from the slope that represents the size bias. This procedure can be readily adapted to other systems where determining the three prime ends of DNA molecules would add key insights into ares of nucleic acid metabolism. Please don't forget that working with HIV can be extremely hazardous and some infections should only be carried out in designated safety laboratories.
