This method can be used to answer key questions in Hepatitis B virology, such as finding cellular or virological factors that affect HPV DNA integration. There are multiple advantages to this technique. It's relatively cheap, and easy to carry out, analysis of the results is simple, and it's very sensitive, down to single copies.
Though this method can be used to provide insight into HBV integration, it can also be used for other viruses that integrate into the host cell genome, such as HIV, HTLV-1, or HPV. To infect the cells, seed 200, 000 cells per milliliter in a twelve well plate, with 1 milliliter of D-mem. On the next day, use Heparin column purified su-per-na-din from HEP-AD 38 as inoculant to infect the cells at 1, 000 VGE per cell, in 500 microliters of culture medium.
Then, culture the cells at 37 degrees Celsius. Then, on the next day, wash the cells twice with one milliliter of sterile one times PBS. Then, replace the culture medium every 2 days, until harvest.
At day 3 post infection, treat the cells with 5 micromolar tenofovir disoproxil and 10 micromolar Lamivudine to limit production of HBV replicative intermediates that are amplify able by inverse PCR. At day 5, post infection, trypsinize some cells with 200 microliters of Trypsin EDTA and re suspend them in 2 milliliters of D-mem containing 5 micromolar Tenofovir disoproxil and 10 micromolar Lamivudine. Subsequently, transfer the cell suspensions to a six well plate, to induce one round of mitosis.
At day 7 post infection, trypsinize the expanded cells in 400 microliters of Trypsin EDTA and suspend the mixture in 1 millileter of D-mem. Then, transfer the suspension in a 1.5 milliliter tube. Pellet the cells by centrifugation at 500 times G, for five minutes.
And remove the Su-per-na-din by aspiration. Extract the DNA from the cell pellet using a DNA extraction kit, as per the manufacturer's instruction. For DNA inversion, allocate 1.5 to 2.5 micrograms of the total DNA extract into a 200 microliter PCR tube.
Next, add the appropriate amount of restriction enzyme master mix to result in a 40 microliter of reaction volume, containing 1 times digestion reaction buffer and 10 units NCO-1HF. Incubate the restriction enzyme reaction in a PCR machine at 37 degrees Celsius for one hour, for optimal digestion efficiency. Then, inactivate the restriction enzyme by incubating it 80 degrees Celsius for 20 minutes.
Transfer the entire restriction enzyme reaction to a 1.5 milliliter tube. Subsequently, add 400 microliters of 1 times T4 DNA ligase buffer and 500 units of T4 DNA ligase, and mix thoroughly. Large reaction volume encourages intramolecular ligation of the digested DNA fragments.
Incubate the ligation reaction at room temperature for 2 hours to ensure complete ligation. After 2 hours, Inactivate the T4 DNA ligase at 70 degrees Celsius for 20 minutes. Then, add 10 microliters of 10 Sodium Dodedecyl Sulfate to ensure complete inactivation of the T4 ligase.
Add Sodium Chloride to a final concentration of 100 millimolar, and Dextran to a final concentration of 90 micrograms per milliliter. Mix by pulse vortexing, and briefly spin down the reaction mix. Next, add 900 microliters of 100 ethanol, and mix by inversion.
Precipitate the DNA at negative 20 degrees Celsius overnight. And pellet the precipitated DNA by centrifugation at 14000 times G for 15 minutes. Afterward, remove the Su-per-na-dine by aspiration.
Wash the pellet with 500 microliters of 70 ethanol. And centrifugation at 14000 times G for 15 minutes. Then, remove the Ethanol by aspiration and air-dry the DNA pellet at room temperature for 20 minutes.
Dissolve the pellet in 20 microliters of water and add 20 microliters of restrict enzyme master mix to result in a 40 microliter reaction volume, containing 1 times digestion reaction buffer, 5 units BSIHK-1, and 5 units of SPH-1HF. Nest, incubate the restriction enzyme reaction in a heat block at 37 degrees Celsius, for 1 hour. Briefly spin down the reaction mix, incubate the restriction enzyme reaction in a heat block at 65 degrees Celsius for 1 hour.
In this procedure, remove potential amplicons from a reusable silicon mat seal, using a DNA degradation solution. Rinse the mat thoroughly with DNA free water and air dry it at room temperature. Next, prepare one millileter of 1 times PCR mix containing the outer forward and reverse primers at a concentration of 0.5 micromolar.
Add 170 microliters if the 1 times PCR mix to wells A-1 and E-1, in a 96 well PCR plate. Then, add 120 microliters of the 1 times PCR mix to wells B-1 and H-1. Afterward, add 10 microliters of the inverted DNA to wells A-1 and E-1.
Mix the reaction in each well, by gently pipetting each, about 10 times using a P-1000 set to 100 microliters. Serially dilute the samples from wells A-1 to D-1, at a ratio of 1:3 by transferring 60 microliters at each step. Mix the wells at each step by gently pipetting them about 10 times using a P-1000 set to 100 microliters.
Avoid forming bubbles, repeat the procedure for well E-1, diluting down to well H-1. Subsequently, allocate 10 microliters of the reaction mixture, using multichannel pipette, from Wells A-1, H-1, into wells A-2, H2, A-3, H-3 and so-on, until reaching wells A-12, H-12 of the 96 well plate. Cover the PCR plate with a dry silicon mat, and press firmly.
Then, place the plate in a PCR machine, and run the program indicated, before transferring the plate to room temperature. Afterward, heat the pins of a 96 pin replicator to red-hot with a Bunsen burner, then cool for at least 5 minutes at room temperature. Fill the wells of a second PCR plate with 10 microliters of 1 times PCR mix, containing a gel load ready buffer, and the inner forward and reverse at 0.5 micromolar.
Carefully remove the silicon mat from the PCR plate of the 96 well plate from the first round PCR, and avoid cross-contamination between wells. Use the cooled replicator to transfer the PCR products of the 96 well plate, from first round PCR, to the newly allocated 96 well plate. Carry out the nested PCR, using the condition as indicated.
To isolate the PCR products, analyze them by gel electrophoresis, using a 96 well plate, with 1.3 Agarose gel. For a 100 milliliter Agarose gel, run at 200 volts for 10 to 15 minutes. Afterward, excise the DNA bands from the Agarose gels, using disposable drinking straws.
For each PCR product, place the straw and Agarose gel plug in a 1.5 milliliter tube, trim the straw to size with scissors. Afterward, eject the Agarose plugs into each tube, by squeezing on the end of the straw fragment. Then add 300 microliters of gel extraction buffer, and 5 microliters of gel extraction glass beads to each tube.
Extract the PCR products per manufacturer's instructions for the gel extraction kit, and dilute the DNA from the beads with 30 microliters of water. Submit the purified DNA for Sanger sequencing, with the forward primer used in the second round nested PCR. An example Agarose gel electrophoresis of successful inverse PCR, before and after PCR product isolation, is shown here.
M represents DNA marker latter, each row of 12 represents the technical replicates at a single dilution on the PCR plate. In this instance, single PCR products should be achieved by the second or third 1:3 dilution of each sample. At these dilutions, approximately 50%of products will represent true virus cell DNA junctions.
While the other half represents amplification of HBV DNA intermediates. This technique has allowed researchers to explore HPV DNA integration in highly controlled in-vitro infection systems. Additional methods, such as biothematics analysis, can be used to answer further questions.
For example, if HPV DNA integration occurs in specific regions in the cellular genome. Don't forget that working with infection Hepatitis B virus, can be hazardous and appropriate infection controls, such as bio safety cabinets and prior vaccination, should always be taken while performing these procedure.
