Periodontal Disease Unrecognized Condition Clinical Trial
Official title:
Design of a Protocol for Obtaining Genomic DNA From Saliva Using Mouthwash: Samples Taken From Patients With Periodontal Disease
Objective The aim of this study was to test a protocol for the extraction of high quality
genomic DNA from saliva samples obtained with mouthwash and taken from patients with
periodontal disease.
Materials and methods Saliva samples were taken from 60 patients, then stored at room
temperature. DNA extraction was carried out at distinct post-sampling times (10, 20 and 30
days). Evaluation of genomic DNA was performed with spectrophotometry, electrophoresis, and
PCR genotyping and sequencing.
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.
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Time Perspective: Prospective