gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The aim of the following experiment is to analyze the sequence of 11 genes simultaneously with a very easy and relatively fast protocol based on transposase based technology. This is achieved by the action of a transposase to obtain 300 base pair DNA fragments directly ligated with adapters. The first capture using specific probes, enriches samples in regions of interest.

This step is repeated once again to obtain only target sequences. Next PCR amplification is performed in order to increase the quantity of materials to sequence results are obtained that show genetic variations in genes based on sequence alignment and bioinformatics analysis. The main advantage of this technique over existing methods like Sanger sequencing, is its great capacity that enable us to analyze 24 passions for 11 complete genes in less than two weeks.

Demonstrating the procedure will be sandy, a technician for my laboratory. To begin, prepare all reagents from the DNA enrichment kit. When ready, add approximately 50 nanograms of genomic DNA into each well of a 96 well plate begin mentation by adding 25 microliters of TD buffer and then five microliters of TDE one buffer.

To each well set a pipette to 50 microliters and gently pipette the entire volume up and down 10 times. Centrifuge briefly, and then place the plate in a thermocycler when ready. Vortex the ST solution and add 15 microliters to each well containing sample.

Set the pipette to 65 microliters and gently pipette the entire volume up and down 10 times incubate for five minutes of room temperature before centrifusion. Next, add 52 microliters of previously mixed purification magnetic beads to each well mix and incubate for 10 minutes of room temperature before centrifuging. After washing the beads with ethanol, remove the plate from the magnetic stand and add 22.5 microliters of RSB buffer.

After mixing, place the plate on the magnetic stand and incubate for two minutes. Ensure that the supernatant appears completely clear. Gently transfer 20 microliters of the snat to a new 96 well plate.

To begin the first round of PCR amplification, add 20 microliters of LP PMM and five microliters each of index one and index two to each. Well mix and cover the plate with an adhesive Micros EAL film. After centrifuging, place the plate in the Thermocycler and Run program one.

After purifying and determining the yield from the first round of PCR, begin the first hybridization by pulling up to 12 samples. In the new 96 well plate ensuring that the final volume of each pool does not exceed 40 microliters thoroughly Mix the NCT one solution and add 50 microliters to each well. After adding 10 microliters of CSO, mix and seal the plate.

After centrifuging, place the plate in a thermocycler and Run program.Two. For the first hybridization wash, remove the plate from the thermocycler and centrifuge. Briefly remove the adhesive cover very carefully.

Transfer 100 microliters of reaction mix from the plate to a new MIDI 96 well plate and add 250 microliters of well Vortex SMB solution. Mix and seal the plate before incubating at room temperature for 30 minutes. After washing and discarding the supena, add 200 microliters of well mixed WS two solution into wells containing beads and mix thoroughly trans transfer the entire volume into a new 96 Well plate.

Seal the plate and place in a thermocycler. Launch the thermocycler at 42 degrees Celsius for 30 minutes. Once complete, immediately place the plate on the magnetic stand for two minutes.

Ensure that the supernatant appears completely clear. Remove the seal and immediately discard all the supernatant. Remove the plate from the magnetic stand.

Add 200 microliters of well mixed Ws three solution into wells containing beads and mixed thoroughly. After washing and removing the supinate, seal the plate and briefly centrifuge to pull down residual supine natant. Place the plate on the magnetic stand for two minutes.

Discard the residual supinate. Next, remove the plate from the magnetic stand. Add 23 microliters of previously prepared mix.

After mixing, seal the plate and leave for five minutes of room temperature before centrifuging. After transferring 21 microliters of sate to a new 96 well plate add four microliters of ET two solution to each. Well mix and seal the plate before centrifusion.

To begin the second hybridization, remove the adhesive film and add 50 microliters of NCT one 10 microliters of CSO and 15 microliters of PCR Grade water to the 25 microliters of library. After centrifuging, place the plate in a thermocycler and run program.Two. In a new 96 well plate mixed 20 microliters of evolution from the previous step.

With 25 microliters of T-C-P-M-M and five microliters of PPC seal the plate after mixing and centrifuge. Next, place the plate in a thermocycler and run program three the following day, centrifuge the plate and wash the beads with ethanol. Remove the plate from the magnetic stand and add 30 microliters of RSB buffer.

Mix and incubate the plate for two minutes. At room temperature, place the 96 well plate on the magnetic stand and incubate for five minutes or until the S supernatant appears completely clear. Gently transfer 28 microliters of the snat into a new 96 well plate.

The final step is to determine the quality of the library by using a fragment analyzer or QPCR. During the run, the cluster density should be between 801, 000 K per millimeter square. Different images correspond to low density, high density, and perfect density.

Results obtained with this technology are very easy to interpret as either point or indel mutations using bioinformatics. The base changing is indicated and the deleted or inserted sequence is directly identified. Once mastered, this technique can be done in less than three days if it's performed properly.