Collection and Extraction of Saliva DNA for Next Generation Sequencing

The overall goal of this procedure is to extract high quality genomic DNA from whole saliva. This is accomplished by first lysing the cells to release the DNA. The second step of the procedure is to remove RNA and protein impurities.

The third step is to precipitate the genomic DNA and remove any remaining contaminants or impurities. The final step is to rehydrate the genomic DNA for downstream applications such as PCR and sequencing. Ultimately, spectro, photometry and amplification can show that the collective genomic DNA is of high quality.

The main advantage of this technique over collection kits is the costs will be significantly reduced without any loss in yield. One half hour before collecting saliva, subjects should rinse out their mouths with water and then refrain from food or drink until the sample is taken. To take the sample, have the subject spit precisely 2.5 milliliters of saliva into a 15 milliliter tube containing 2.5 milliliters of DNA stabilization buffer.

Do not touch the inside of the tube or the inside of the cap. During this process, cap the tube and homogenize the mixture with inversions not shaking. Then store the sample at room temperature for the short term or at four degrees Celsius for up to three months.

Preparations include heating a water bath to 37 degrees Celsius and having three 15 milliliter tubes at the ready. Begin by inverting the sample a few times, then vortex it at a medium speed for 15 seconds. Transfer 2.5 milliliters of the sample to one of the tubes.

Add five milliliters of cell lysis solution to the aliquot and mix the lysis solution into the sample by inverting the tube 50 times. Once the sample is mixed, let it incubate for 30 minutes. At room temperature after half an hour, add 40 microliters of RNA A at 100 milligrams per milliliter to the lysed cell solution.

Then quickly invert the tube five times. Let the RNA reaction incubate at 37 degrees Celsius for 15 minutes. Then cool the tube on ice for three minutes.

At this point, increase the water bath temperature to 65 degrees Celsius. Next, remove the tube from the ice. Add 50 microliters of proteinase K and mix either let the reaction incubate at room temperature for 30 minutes or 16 to 24 hours at four degrees Celsius.

Later, add 1.7 milliliters of protein precipitation solution, and vortex the tube for 20 seconds at high speed. Then let the sample cool on ice for 10 minutes. Once cooled, centrifuge a sample for 10 minutes at 3000 Gs and at four degrees Celsius.

If the pellet is not tight, cool the sample for five more minutes and centrifuge it again for five minutes. Repeat this process until a tight palate is obtained. Always keeping the sample cold.

Begin the DNA isolation by preparing a clean 15 milliliter tube with five milliliters of isopropanol and eight microliters of glycogen solution at 20 milligrams per milliliter. Then pour the supine agent of the spun down sample into this tube. Discard the pellet, mixed a sample 50 times by inversion.

Next, spin down the sample at 3000 Gs and at four degrees Celsius for 30 minutes. Pour the supinate into another clean 15 milliliter tube to the pellet. Add one milliliter of 70%ethanol and rock by hand on its side for 30 seconds.

Then centrifuge the ethanol solution for a minute at 2000 Gs and at either four degrees Celsius or 20 degrees Celsius. Discard the sennet and by slowly pouring it off, then add more ethanol to the pellet. Rock the two by hand and repeat the spin.

Then slowly pour off the ethanol again. During the wash, the pellet may become loose. If it detaches, make sure it stays in the ethanol solution and does not stick to the sides or lid of the tube.

Do not invert the tube during this wash. After the ethanol washes, set the tube to air dry for 15 to 30 minutes. Then rehydrate the pellet of genomic DNA by first adding 300 microliters of tris EDTA.

Then vortex a sample at medium speed for five seconds and transfer the sample to a 65 degree Celsius water bath for an hour after being heated in the bath, let the sample incubate overnight at room temperature the next day the DNA will be ready for use gel electrophoresis of saliva, DNA extraction samples show appropriately. High molecular weight bands without the smearing indicative of degraded DNA. The RNA removal step is necessary and very effective.

After the RNAs a digestion, the 260 nanometer to 280 nanometer ratio averages 1.74. Importantly, the DNA is suitable for high throughput sequencing. A target enrichment kit was applied to 24 samples targeting 2.6 megabases of DNA 12 barcoded samples were sequenced per lane.

All 24 samples had high quality NGS data and rare single nucleotide polymorphisms could be identified. After watching this video, you should have a good understanding of how to extract high quality genomic DNA from whole saliva.