The overall goal of this procedure is to infer viral tropism in an HIV infected patient by sequencing the V three region of the viral envelope gene to begin extract H-I-V-R-N-A from patient's blood plasma. Next, amplify the V three region of HIV GP one 20 using nested R-T-P-C-R. Then identify the presence of product using gel electrophoresis, followed by population-based sequencing of the amplified V three products.
Finally analyze the sequences using base calling software and infer co-receptor usage using the genome to pheno algorithm. Ultimately, these results can show whether a patient's viral troop is R five or non R five through population-based sequencing of the HIVV three loop. So the primary advantage of this technique is a troop assay over existing methods such as phenotypic cell-based assays is that it can be performed in any laboratory equipped with sequencing apparatus for reduced cost in less amount of time with less starting material.
This technique is relevant to treatment with antiretroviral therapy because it can identify whether a patient's viral population is likely to respond to a CCR five antagonist From 500 microliters of plasma. Use an easy mag automated extractor to extract H-I-V-R-N-A. Now move to A PCR clean room to set up the R-T-P-C-R in order to ensure adequate sampling of the viral population.
Plan to perform each reverse transcription reaction in triplicate in this one step reaction. Use viral RNA to synthesize CDNA, specifically amplifying the HIVV three region with the SQV three F1 and CO 6 0 2 prime repair. Based on per reaction components.
Make a stock mix solution sufficient for the number of samples to be amplified using a repeater pipette Add 36 microliters of sample mix to each well of the PCR plate. Now switch to a multi-channel pipette to transform microliters of RNA sample extract to each of designated wells. Take care to change tips between each addition.
Once the transfer is complete, cover the wells with PCR strip caps. Then place the PCR plate in a thermocycler programmed for a one step R-T-P-C-R of the HIVV three region. For the nested PCR, use the SQV three F two and CD four R primer repair.
Calculate the amount of reagent required for the number of samples to be amplified in a PCR clean room. Generate the nested PCR mix and then use a repeater pipette to aliquot 19 microliters per well of a clean PCR plate. On completion of the R-T-P-C-R, move to a post amplification room.
Use an eight channel multi-channel pipette to transfer one microliter of the R-T-P-C-R template into the nested PCR mix. Change tips between each addition. Cover the wells with PCR strip caps and transfer the plate to a thermocycler for amplification.
Remove the plate from the thermocycler after the temperature has returned to 25 degrees Celsius. In order to confirm that the V three region has been successfully amplified, first analyze products by gel electrophoresis. Prepare a 1.6%AROS gel with cyber safe gel stain.
Using a 10 microliter multichannel pipette. Transfer eight microliter PCR samples into respective wells. Also load a DNA ladder to at least one well per row.
Run the gel at around 100 volts for 10 minutes. Visualize bands by ultraviolet fluorescence and document the image by photography. Make note of the samples where all triplicate amplifications were successful.
If necessary, repeat R-T-P-C-R for those samples with fewer than three product amplifications. The amplified viral CDNA samples are further verified by DNA sequencing reactions will be performed using the V 3 0 2 F and S QV three R one primers. Remove the big di reagent from the freezer and thaw at room temperature.
Calculate and prepare the reagents required for the number of samples to be run. Aliquot five microliters of sequencing reaction mix into the appropriate plate wells using a multi-channel pipette. Cover the plate with a Kim wipe and set it aside.
Meanwhile, prepare a one to 15 dilution of the amplified PCR product by adding 180 microliters of sterile water to the remaining PCR product. Pipette one microliter of the diluted sample to the bottom of the appropriate plate. Wells always change tips between samples when the sample transfer is complete.
Seal the plate with strip caps and vortex for one second. To centrifuge the plate, set the speed to 140 5G. When the spin speed reaches 140 5G, stop the spin.
Place the plate in a thermocycler for the reaction to proceed. In each well precipitate the DNA with four microliters of three molar sodium acetate and 40 microliters of chilled 95%Ethanol, reseal the caps. Cortex the plate for 10 seconds and tap the plate to dislodge any air bubbles.
Place the plates at minus 20 degrees Celsius for between 30 minutes and two hours. Recover DNA by centrifugation at 2000 G for 20 minutes. Remove and discard the strip caps.
Invert the plate over the waste container and decant the super natin. Rid any remaining super natin. By blotting the inverted plates on a paper towel.
Fold two sheets of a paper towel to sandwich the plate. Place the plate face down in the centrifuge and spin at 140 5G. Stopping the spin when it reaches 140 5G.
Then wash with 155 microliters of 95%ethanol and decant. Then repeat the centrifugation. Let the plate sit for two to five minutes to allow any last ethanol to evaporate.
In order to denature the DNA samples, use a multi-channel pipette to distribute 10 microliters of high dye for maide into all wells on the plate, including those that are empty. Seal the plate with a septima and place in a thermocycler at 90 degrees Celsius for two minutes. Finally, place the sample plate into a sequencing plate and into the sequencer.
One acceptable route to data analysis is use of the software recall to perform automatic base calling with no human intervention. Recall is a free custom-based calling software log in and select sample files to upload with the browse option. Locate the raw sequencing data file and upload up to 20 megabytes.
Next, set the reference sequence to V three. Click process data. Select the resulting folder on the right side of the page to show the list of samples.
Those that are red have failed. Those that are green have passed. By clicking on a specific sample and the view button, you are able to review the sequence, follow the instructions on the right side of the page to navigate through the sequence, download files into a zip folder.
Viral tropism can be inferred from sequences generated by population-based sequencing using the geno to pheno co-receptor algorithm. From the dropdown menu of the significant setting, select the optimized based on analysis of clinical data from motivate 2%and 5.75%FPR. Now choose to upload or paste a sequence for analysis.
FASTA files are acceptable. Do not add other proceed to obtain a geno Tono FPRA predictor of responsiveness to CCR five antagonists. The G two P report generated provides the V three loop sequence in amino acids and indicates relevant positions from mutations associated with non R five using virus.
At the bottom of the report, the false positive rate is given and interpreted in terms of response to a CCR five antagonist. This report can be downloaded as a PDF file. If any one of the three sequences from a sample is inferred to be non R five, the patient is not likely to respond to a CCR five antagonist as expected gel electrophoresis of RTP CR Amplifications of the HIV one V three loop indicates that not all clinical samples produce a product.
The generated sequences from the amplified samples can be visualized in a chromatogram read by recall. A clean sequence with few mixtures is expected. Occasionally sequences may be messy and not amenable to accurate base assignment.
Some patients may be identified as having non CCR five using virus as shown here. These patients are not likely to respond to treatment with a CCR five antagonist. However, the majority of patients are found to have a viral population composed of CCR five using virus as indicated by a green box at the bottom of the G two P analysis report.
So once mastered, this technique can be performed in roughly four days from beginning to end. That's from extraction to analysis. After watching this video, you should have a good understanding of how to predict viral tropism using population-based sequencing methods in conjunction with the genome pheno bioinformatic algorithm.
All right, that's it. Thank you for watching and good luck with your experiments.
