The overall goal of the following experiment is to identify the exact positions of integrating viral vectors in the genome. This is achieved by an initial linear PCR reaction with biotinylated primers to enrich for vector genome junction sequences from genomic DNA on solid phase in a series of reactions, double stranded DNA with known DNA sequences on both ends of the product are generated, which allows for exponential amplification of vector genome junctions. Next sequencing adapters are incorporated to lamb PCR products.
In order to sequence and characterize the unknown flanking DNA with deep sequencing, these fragments reveal the exact positions of vector integrations. The result is a diversity of amplified junctions as viewed by gel electrophoresis. This method provides insight into viral vector integration site distribution in clinical gene therapy patients.
It can also be applied to other studies such as insertional, mutagenesis, screens, virus infection, cancer, and stem cell clonality or T-cell diversity. The procedure will be demonstrated by ina RA, a technician from my laboratory For this procedure, first prepare the linker cassettes mix 40 microliters of each of the two LCO Legos with 110 microliters of tris hydrochloride, and 10 microliters of magnesium chloride in a micro centrifuge tube. Next, set the mixture to incubate on a 95 degrees Celsius heat block for five minutes, and then cool slowly to room temperature overnight.
The next day, add 300 microliters of water to the prepared double stranded linker, DNA, and filter them over a micro centrifuge tube. Spin down the filter to collect the concentrated linker DNA in the ouit. Next, recover the ouit from the filter.
Add 80 microliters of water to the ouit and pipette. 10 microliter aliquots into PCR tubes. Use PCR to pre amplify the vector genome junctions in 50 microliter reaction tubes for lamb.
Add between one nanogram and one microgram of sample DNA per 50 microliter reaction. For NR lamb, use at least 100 nanograms of DNA. Do not add more than 25 microliters of execute the PCR according to table two in the text.
And when completed, add an additional 0.5 microliters of tech to each reaction and run the PCR program a second time. First, prepare the magnetic beads. Add 20 microliters of strept ENC coated magnetic beads to a 1.5 milliliter tube and put them on the magnetic beads separator for one minute at room temperature.
Then discard the supernatant raise. Suspend the beads in 40 microliters of PBS with BSA and put the beads back on the separator for another minute. Replace the supernatant with a 20 microliter wash of three molar lithium chloride solution.
And again, put the beads on the separator for a minute. Finish by replacing the supernatant with 50 microliters of six molar lithium chloride solution. Now add the magnetic bead mix to the product of the PCR reaction and allow this mixture to incubate for two hours to overnight at room temperature with gentle shaking.
The biotinylated DNA and strip tava and coated beads will form complexes that can be stored at four degrees Celsius for up to four days. Proceed with lamb or NR lamb Due to its high sensitivity. L-A-M-P-C-R is prone to contamination if executed in attentively.
That's a PCR grade environment. And special attention to cleanliness of utmost importance. To successfully amplify the unknown flanking DNA without contaminating the samples, First expose the complexes to the separator for a minute and resus suspend the DNA beads in 100 microliters of water.
Next, expose the complexes to the separator for a minute. And this time resuspend the DNA bead complexes in a mixture with 8.25 microliters of water. One microliter of HEXA nucleotide buffer, a quarter microliter of D DN NTPs, and half a microliter of cano polymerase.
Incubate this mixture at 37 degrees Celsius for an hour. Then add 90 microliters of water and expose the reaction to the separator for another minute. Then resuspend the DNA bead complexes in a 100 microliter wash of water.
Use the separator for another minute and prepare the restriction enzyme reaction. After removing the supernatant. Add 8.5 microliters water, one microliter of restriction enzyme buffer, and half a microliter of the restriction enzyme.
When selecting the restriction enzyme, make sure that it has no sites in or downstream of the primal binding site used in the preem amplification reaction. After allowing the enzymes to react for an hour at the ideal temperature, add 90 microliters of water. Use a separator for a minute and resuspend the BDNA complexes in a wash of 100 microliters of water.
Repeat the separation and prepare the ligation reaction. Add to the beads five microliters of water, one microliter of 10 x fast link buffer. One microliter of a TP one microliter of fast link DNA ligase and a microliter of the linker cassette made at the beginning of the procedure.
Allow this reaction to run for five minutes at room temperature. End the reaction by adding 90 microliters of water and separating the beads, followed by a wash in 100 microliters of water. And then another separation to denature the synthesized DNA resuspend the beads in five microliters of 0.1 normal sodium hydroxide, and allow the mixture to shake for five minutes at room temperature.
Then separate out the complexes for a minute and collect the SUP natin, which contains the preem amplified vector genome junction. Immediately proceed with the exponential amplification or store at minus 20 degrees Celsius. Begin with exposing the complexes to the separator for a minute.
And resus suspending the DNA beads in 100 microliters of water. And separate them again and remove the supernatant. For the ligation reaction, add 6.5 microliters of water, one microliter of circ Ligase, 10 x reaction buffer.
Half a microliter of magnesium chloride, half a microliter of a TP, one microliter of SS linker oligos, and half a microliter of cir ligase. After an hour at 60 degrees Celsius, end the reaction with 90 microliters of water using the bead separator Resus suspending the beads in another 100 microliters of one, using the separator again and collecting the wash complexes in 10 microliters of water. Begin by preparing a PCR master mix as described in table three of the text.
Add 48 microliters of master mix to two microliters of lamb or NR lamb products in 200 microliter PCR tubes and run the PCR as described in the text as before, prepare more strept in beads and resuspend them in six molar lithium chloride. Use 20 microliters for lamb or 50 microliters for NR lamb. Then add beads to the same volume of PCR reaction products and incubate them on a shaker at 300 RPM for two hours to overnight at room temperature, collect the DNA bead complex and wash it with 100 microliters of water as previously demonstrated.
Now, suspend the complex in 0.1 normal sodium hydroxide and incubate the beads for 10 minutes at room temperature on a shaker. Then expose the beads to the separator for a minute and collect the supernatant containing the amplified DNA to a new tube. Using the amplified DNA as a template, use two microliters of it to run a PCR as described in table four of the text.
Visualize the products by gel electrophoresis to purify the products. Mix 40 microliters of them with 44 microliters of room temperature am pure XP magnetic beads, and incubate them for five minutes. Then separate the beads for two minutes on the magnet.
After removing the supernatant, wash the beads twice using 200 microliters of 70%ethanol, and then resuspend the beads and 30 microliters of water using 40 nanograms of DNAA fusion primer. PCR can be used to add sequencing specific adapters. Details are given in table five.
Check the products on a gel. Viewing the results of lamb PCR by high resolution gel electrophoresis is the best choice of diagnostic analysis by comparison. While 2%aros also works, it does not work as well.
Clonality of NR LAMB, PCR products cannot be diagnosed visually. A 2%agros gel is however perfectly sufficient to determine the success of the protocol. After sequencing the PCR products, the location of the vectors can be found in the host genome.
From this data, it is possible to log insertion site locations in coding regions, and close to transcription start sites After its development. This technique paved the way for researchers in the field of gene therapy to explore the biosafety of gene transfer vectors in both preclinical models as well as in clinical gene therapy trials.
