Saliva Preservation Buffer

Genomic DNA Isolation

DNA Stabilization Buffer (250 ml)

Component	Volume (ml)	[Final]
1 M NaCl	1.461 g	0.1 M
Tris HCl	2.5	0.01 M
0.5M EDTA	5	0.01 M
10% SDS	12.5	0.014 M
Proteinase K Solution (20 mg/ml)	2.5	6.92x10-6 M
ddH2O	227.5

1. Saliva Collection and Storage

Prior to saliva collection, ensure that the subject’s mouth is free of food or other foreign substances by having the subject rinse their mouth with water and avoiding eating or drinking for 30 min before collecting the sample.

Open a 15 ml centrifuge tube with 2.5 ml of DNA stabilization buffer making sure to avoid touching the inside of the cap or tube, and have the subject spit 2.5 ml of saliva into the buffer solution.

Note: Collecting more than 2.5 ml of saliva can lead to sample degradation from an insufficient ratio of sample to DNA stabilization buffer. Collecting too little saliva will reduce expected yields from the protocol. To evaluate collection volumes, use the numbered gradients on the side of the tube.

Replace cap and mix by inversion until the mixture is homogenized. Vigorous shaking is not necessary.Store the samples at RT for short-term storage or 4 °C for long-term storage (>3 months).

2. Initial Preparations and Cell Lysis

Prior to starting the extraction, heat a water bath to 37 °C, and prepare an ice bucket. Three 15 ml conical centrifuge tubes will be needed for each extracted sample. The three tubes will be used to hold the cell and protein pellet, the final extracted gDNA, and the isopropanol and ethanol supernatants.

Retrieve samples from storage, and invert samples several times then vortex at medium speed for 15 sec.

Dispense 2.5 ml of sample into a clean 15 ml centrifuge tube, and add 5 ml of Cell Lysis Solution. Mix the sample 50 times by inversion, and incubate at RT for 30 min.

3. RNA Removal

Add 40 μl of RNase A Solution at 100 mg/ml, and incubate at 37 °C for 15 min.

Remove the sample from the 37 °C water bath and cool on ice for 3 min.

After the RNase A incubation, increase the temperature of the water bath to 65 °C for the DNA rehydration step of the protocol.

4. Protein and Lipid Removal

Add 50 μl of Proteinase K Solution at 20 mg/ml, mix several times by inversion, and incubate at RT for a minimum of 30 min. Note: This is a possible pausing point for the protocol. After the addition of the Proteinase K Solution, the sample can be stored at 4 °C until the extraction can be completed. Storage at 4 °C for up to 24 hr was not shown to have a significant effect on the extraction yields or DNA quality. Long-term storage at this stage has not been evaluated.

Add 1.7 ml of Protein Precipitation Solution, vortex vigorously for 20 sec at high speed, and place on ice for 10 min.

Once the samples have cooled on ice for 10 min, centrifuge for 10 min at 3,000 x g and 4 °C. The precipitated proteins must form a tight pellet to continue. If the pellet is not tight or the solution is still cloudy, the samples can be cooled on ice for 5 min more and centrifugation repeated. The samples must be kept on ice to ensure a tight pellet.

5. Isolation and Purification of gDNA

Into a clean 15 ml centrifuge tube, pipet 5 ml of Isopropanol and8 μl of pure Glycogen Solution at 20 mg/ml.

Pour the supernatant containing the gDNA from step 4.3 into the tube containing the Isopropanol and Glycogen Solution, leaving behind the precipitated protein pellet. Once the supernatant has been added, gently mix the sample 50 times by inversion and centrifuge for 30 min at 3,000 x g and 4 °C.

Pour the supernatant slowly into a clean 15 ml tube. After removal of the supernatant, add 1 ml of 70% ethanol to wash the pellet by slowly rocking and gently moving the ethanol over the precipitated pellet several times. Retain the ethanol in the tube.

After the initial wash, centrifuge the sample for 1 min at 2,000 x g and 20 °C. This centrifugation step can be done at either 4 °C or 20 °C. No significant effect of temperature has been shown for this step.

Following the initial wash and centrifugation of the pellet, slowly pour the ethanol wash from the tube and discard, then perform a second wash by repeating steps 5.3 and 5.4.

After the removal of the supernatant from the second wash, allow the pellet to air dry for 15 min.

If the sample has not completely dried, air dry for another 15 min.

6. Rehydration of gDNA

Once the sample has dried, add 300 μl of Tris-EDTA to rehydrate the dried gDNA pellet.

Vortex the sample for 5 sec at medium speed and place in a 65 °C hot water bath for 1 hr.

Remove the samples from the water bath and incubate O/N at RT.

Ref https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442313/

Phenol-Chloroform Protocol

Prepared saliva samples were centrifuged at 12,851 g for 10  min  and  pellets  were  resuspended  in  200 μL  of  SE  buffer  (75 mM NaCl; 25 mM EDTA, pH 8.0). 35  μL  of 20  mg/mL  proteinase  K  and  10 μL  of  10% SDS  were  added.  The  samples were digested at 55 °C for 3 h and DNA was extracted with equal  volumes  of  phenol-chloroform  (1:1).  DNA  was  precipitated  from  the  supernatant  with  3  M  sodium  acetate  (pH  6.0;  volume  40  μL) and two volumes of cold 100 %  ethanol and a following incubation at  –20°C for 15 – 30 min. The DNA was pelleted at 20,817 gfor 25 min, and dried at 37 ° C. The pellet was resuspended in 30 μL redistilled water at 55 ° C for 30 min. 

Qiagen Protocol

For the cytobrush protocol, 400 μL PBS were added to the cytobrush in the cryotube. After vortexing for 15 seconds and incubating at 56◦C for 15 minutes 400 μL ethanol (96%–100%) were added and mixed by vortexing. For the mouthwash protocol, cells were pelleted by centrifuging the 50-mL tube for 10minutes at 1 500 g. The pellet was resuspended in 200 μL of supernatant. The DNA was eluted with 150 μL buffer AE into a clean 1.5mL microfuge tube for cytobrushes and 200 μL for mouthwash. For treated card protocol, DNA was extracted according to the protocol of the manufacturer (Schleicher & Schuell) and template for amplification was contained in the 100 μL eluate

Clinical Trail

https://clinicaltrials.gov/ct2/show/study/NCT02523326?show_desc=Y#desc

Materials and methods 2.1. Patients The study was conducted with 60 patients diagnosed with severe chronic periodontitis in the Periodontic Clinic of the Interdisciplinary Health Sciences Center (Santo Tomás Unit), National Polytechnic Institute. The protocol of this study was approved by the Research and Bioethics Committee of the School of Medicine. National Polytechnic Institute. Informed consent was obtained from all participants included in the study, who signed the appropriate form allowing for the collection of saliva samples and the extraction of genomic DNA. Ethical norms for research on human beings, established by the Declaration of Helsinki (1975; updated most recently in 2008) were strictly followed. Each patient was instructed how to prepare for saliva sampling, which involved brushing their teeth at least 2 h previously and abstaining from eating or drinking anything.

2.2. Taking the sample In a tube of 50 ml were placed 10 ml mouthwash (Listerine Cool Mint, Johnson & Johnson, 21.6% alcohol). Patients were instructed to rinse their mouth vigorously with this quantity of mouthwash during 30 s, then spit it into the tube. Once collected, the samples were stored at room temperature (20°C - 24°C). The 60 samples were divided into 3 groups (n=20), and the extraction of DNA was performed at distinct times: group A at 10 days, group B at 20 days and group C at 30 days.

2.3. DNA extraction To separate the mouthwash, the tube was centrifuged during 10 min at 10750x g. The supernatant was poured out and the cellular pellet washed with 1 ml of PBS, resuspended in the vortex for 30 s, and transferred to a new 2 ml tube. It was then centrifuged for 5 min at 2000x g, the supernatant poured out, and the pellet washed with 1.5 ml of PBS and resuspended in the vortex for 30 s. To this solution was added a 1 ml solution of lysis of nucleated cells (Wizard® Genomic DNA Purification Kit) before being shaken for 20 s, followed by incubation at 37 °C for 10 min. After adding 300 µl of protein precipitator solution (Wizard® Genomic DNA Purification Kit), the mixture was placed on ice for 5 min and then centrifuged at 15000x g for 3 min. The supernatant was put in a 1.5 ml tube containing 600 µL cold Isopropanol and homogenized gently, then centrifuged at 15000x for 3 min. The supernatant was poured out and 600 µL of 70% ethanol were added to a fresh preparation, which was centrifuged at 15000x g for 3 min. The supernatant was poured out and the tube was completely dried at room temperature. Finally, 100 µL rehydration solution (Wizard® Genomic DNA Purification Kit) was added and the mixture was incubated at 64 °C for 1 h before storing the samples at -20 °C to await further processing.

2.4. DNA evaluation Evaluation of the concentration and purity of DNA was determined by spectrophotometry with an ACT-GENE ASP-3700 spectrometer. The concentration of DNA was evaluated at 260 nm, while purity was estimated with the ratios of 260/203 and 260/280. The integrity of genomic DNA was assessed in a 0.8% agarose gel in 1% TBE (89 mM Tris Borate, 89 mM boric acid, 2 mM EDTA) by electrophoresis, and then the DNA was visualized with Gel-Red and read under UV light. DNA extracted from blood samples was used as the positive control.

2.5. PCR The PCR reaction was carried out in an Eppendorf Mastercycler® Personal apparatus with the HotStarTaq ® Master Mix Qiagen kit (No. de Cat 203445), following the manufacturer's instructions. To verify the quality and species (human) of the DNA extracted, primers were designed for the rs679620 polymorphism (forward 5´- ggg gCT TAA ggC ACA TgA gT-3; reverse 5´- ACT TCg ggA TgC CAg gAA Ag - 3´; Tm: 56°C, 40 cycles) that amplified a product of 485 bp. The final PCR product was evaluated with a 2% agarose gel. DNA extracted from blood samples was used as a positive control.

2.6. Sequencing To further verify the quality and species of the DNA obtained, the product resulting from PCR was sequenced with an Applied Biosystems 3130 sequencer and the BigDye® Terminator v3.1 Cycle Sequencing kit, following the manufacturer's instructions. Data were analyzed with Geneious version 7.1.3 software [23], using the NC_000011.10 sequence as the reference.