Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

The overall goal of this procedure is to identify retroviral vector integration sites in the host genome and quantify the relative frequencies of clonal cell populations each sharing the same integration site. This method can help answer key questions in retroviral gene therapy, such as which part of the host genome vector has integrated, how a genetically engineered cell will repopulate after the transplant, and how they are functionally different. The main advantage of this technique is that retro-integration site sequences can be analyzed with higher accuracy and sensitivity by sequencing both upstream and downstream vector host DNA junctions simultaneously.

Though this method can provide insight into the safety of vectors and gene engineered cell in gene therapy studies, it can also be applied to other basic biology studies to investigate individual cell behavior in vivo. The first step in this procedure is to determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers of the genomic DNA to be analyzed. After this, dilute one to two micrograms of the sample genomic DNA to a final volume of 170 microliters of genomic DNA solution using nuclease free water.

Prepare a 200 microliter PCR reaction by mixing the following in a microcentrifuge tube. 2.5 microliters each of HIV-1 specific biotin primers, or a total of 10 microliters for four lentivirus-specific primers, 20 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 millimolar dNTPs, three microliters of thermostable DNA polymerase, and 163 microliters of genomic DNA. Divide the solution equally into four PCR tubes and carry out a single extension cycle under the following condition.

94 degrees Celsius for 5 minutes, 56 degrees Celsius for 3 minutes, 72 degrees Celsius for 5 minutes, and 4 degrees C for storage. Pool all four PCR reactions into a two milliliter microcentrifuge tube. Follow the manufacturer's procedure of a PCR purification kit, and elute with 50 microliters of elution buffer.

Prepare for a 100 microliter digestion by adding the following to a microcentrifuge tube. 50 microliters of DNA, 10 microliters of 10X buffer A, two microliters of RSAI enzyme, and 38 microliters of nuclease-free water. Incubate at 37 degrees Celsius in a thermal cycler for one hour.

Next, add one microliter of CviQI enzyme to the reaction and incubate at 25 degrees Celsius for 30 minutes. When the RSAI and CviQI digestion is complete, proceed immediately with blunt ending. Prepare a 4.5 microliter mixture containing 2.5 microliters of DNA polymerase I, Large Fragment, and 2 microliters of 10 millimolar dNTPs.

Transfer 1 microliter of the mixture to the DNA sample. The total volume will be 102 microliters. Mix well by vortexing and incubate at 25 degrees Celsius for one hour.

Immediately proceed with streptavidin bead binding when the incubation is complete. Prepare the streptavidin beads by briefly vortexing the bead solution and transferring 50 microliters to a new two milliliter microcentrifuge tube. Remove the supernatant using a magnetic stand and wash the beans with 200 microliters of binding solution.

Resuspend the beads in 100 microliters of binding solution and place the tube away from the magnetic stand. Transfer 100 microliters of the sample DNA to the 100 microliters of resuspended bead solution and mix carefully by pipetting to avoid any foaming of the solution. Incubate the tube at room temperature for 3 hours on a rotating wheel.

Use the magnetic stand to capture the beads and discard the supernatant. Wash the beads twice in 400 microliters of washing solution and twice in 400 microliters of 1X T4 DNA ligase buffer. Resuspend the beads in 200 microliters of 1X T4 DNA ligase buffer and place it away from the magnetic stand.

Linker ligation should be performed immediately after the streptavidin bead binding. Prepare a 400 microliter ligation reaction solution by mixing the following in a two milliliter microcentrifuge tube. 0.5 microliters of the DNA linker, 10 microliters of 10X T4 DNA ligase buffer, 20 microliters of 5X T4 DNA ligase buffer, 5 microliters of T4 DNA ligase, 164.5 microliters of nuclease free water, and 200 microliters of the resuspended beads.

Place the reaction tube on a rotating wheel at room temperature for 3 hours. Wash the beads twice with the washing solution, and twice with 1X thermostable DNA polymerase buffer. Resuspend the beads in 50 microliters of 1X thermostable DNA polymerase PCR buffer, and place it away from the magnetic stand.

To begin this procedure prepare a 200 microliter PCR reaction by adding the following to the resuspended beads. 10 microliters of one 1L-primer, 10 microliters of 1R-primer, 20 microliters of primer Link1, 10 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 milimolar dNTPs, eight microliters of thermostable DNA polymerase, and 88 microliters of nuclease-free water. Aliquot the reaction mixture into four PCR tubes and carry out PCR with the following condition.

94 degrees Celsius for 2 minutes, 25 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Pool all four PCR reactions into a 2 ml microcentrifuge tube. Follow the procedure of the PCR purification kit and elute with 50 microliters of elution buffer.

Determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers using a UV visible spectrophotometer. The DNA can now be stored at minus 20 degrees Celsius until ready for the next step. Since the procedures for the left junction and right junction specific amplifications are identical except for the primers used in the PCR, only the left junction amplification will be demonstrated.

Prepare a 50 microliter PCR reaction by mixing the following in a PCR tube. Five microliters of DNA, five microliters of 10 micromolar 2L-primer, five microliters of 10 micromolar primer Link2, 5 microliters of 10X thermostable DNA polymerase buffer, one microliter of 10 milimolar dNTPs, two microliters of thermostable DNA polymerase, and 27 microliters of nuclease-free water. Carry out PCR with the following condition.

94 degrees Celsius for 3 minutes, 8 to 15 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Use the PCR purification kit to purify the DNA and elute with 50 microliters of elution buffer. Determine the DNA concentration and ratio of absorbance at 260 and 280 nanometers.

The last step in generating left and right junction sequence libraries is to analyze the PCR Amplicon length variations by performing capillary electrophoresis. This representative result shows varying lengths of PCR Amplicons in lentiviral vector integrations sites for both the upstream and downstream vector host junctions. The DNA size marker is in the M lane.

After sequencing the PCR Amplicons, computational integration site sequence analysis is performed using an in-house programming script. The bi-directional integration site assay generates different sizes of PCR Amplicons for both the upstream and downstream vector host junctions visualized by capillary electrophoresis. A comparison of percent integration in genes Alu and L1 repeats of lentiviral vectors in humanized mouse repopulating cells within silico generated random integration events revealed that integration sites are significantly enriched in genes.

The relative detection frequencies of vector integromes were calculated using only the sequence counts of integration site junctions generating smaller than 500bp PCR Amplicons. Approximately 77%of the vectors could be quantitatively analyzed by the bi-directional approach. In the strategy for clonal quantification we presume that each individual clone shares the same vector integrome.

The relative frequencies of the left and right junctions are combined to represent the relative quantities of the clonal populations, or QVI. This color scheme shows the relative frequencies of 44 QVI clones in humanized mouse repopulating cells. Based on in silico 10, 000 random integration analysis, a 1.25 fold overestimation of clonal frequencies is expected when using GTAC motif enzymes, and a 2.56 overestimation is expected when using a TCGA motif enzyme.

Once mastered, this technique can be divided in parts and completed in two days, if performed properly. While attempting this procedure it is important to remember that PCR steps are extremely sensitive, and precautions should be take to avoid any contamination. This technique has paved the way to examining the safety of therapeutic retroviral vectors, and to explore long-term repopulation patterns of hemotopoietic stem cell clones following transplant in rhesus macaques.