Collection and Extraction of Saliva DNA for Next Generation Sequencing

Summary

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Abstract

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

Introduction

Obtaining high quality DNA for human genetic studies is essential in the disease gene discovery process. Blood, though requiring an invasive procedure and also being more expensive than saliva collection, is favored for creating immortalized cell lines as an infinite source of DNA, or iPSCs for functional studies, and sometimes blood DNA is used when cell lines are not available. However, obtaining blood requires a trained phlebotomist and blood has a shorter half-life than saliva¹. DNA from saliva is less expensive and easier to obtain, since it can be collected and sent through the mail without the need for a phlebotomist, thereby increasing potential subject pools well beyond the catchment area of hospitals and laboratories². Study enrollment may be improved when subjects have the option of giving a saliva sample instead of blood^{3, 4}. Concerns about the quantity and quality of DNA from saliva may have limited its widespread use despite numerous studies recent studies showing the suitability of whole saliva, with an average of 4.3 x 10⁵ cells per milliliter, for DNA testing over the older buccal swabs methods that did not obtain significant amounts of saliva^{2, 3, 4, 5, 6}. While a modest literature exists showing the suitability of whole saliva derived DNA for genotyping applications including microarray-based methods^{8, 9, 10}, no studies have examined next generation sequencing (NGS). The goal for optimizing this whole saliva DNA extraction protocol was to maximize quantity and quality for genetics applications in a cost effective way that is easily implemented in laboratories with common reagents and consumables.

DNA extraction from saliva requires several procedures: 1) collection and storage, 2) cell lysis, 3) RNase treatment, 4) protein precipitation, 5) ethanol precipitation, 6) DNA rehydration. The DNA Stabilization Buffer solution, described previously², functions adequately without alteration. No attempt to optimize the RNase treatment and DNA rehydration steps was made. For each remaining step, several variables that could affect yield were identified. Each variable was manipulated individually and improvement in yield and quality was assessed statistically. For variables that were shown to improve yield and/or DNA quality, the optimal values were included in the final protocol.

Protocol

NOTE: Prior to providing saliva samples all subjects gave informed consent conforming to the guidelines for treatment of human subjects at Nationwide Children’s Hospital.

1. Saliva Collection and Storage

1. 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.
2. Open a 15 ml centrifuge tube with 2.5 ml of DNA stabilization buffer² 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.
3. 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

1. 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.
2. Retrieve samples from storage, and invert samples several times then vortex at medium speed for 15 sec.
3. 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

1. Add 40 μl of RNase A Solution at 100 mg/ml, and incubate at 37 °C for 15 min.
2. Remove the sample from the 37 °C water bath and cool on ice for 3 min.
   1. 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

1. 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.
2. Add 1.7 ml of Protein Precipitation Solution, vortex vigorously for 20 sec at high speed, and place on ice for 10 min.
3. 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

1. Into a clean 15 ml centrifuge tube, pipet 5 ml of Isopropanol and 8 μl of pure Glycogen Solution at 20 mg/ml.
2. 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.
3. 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.
4. 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.
5. 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.
6. After the removal of the supernatant from the second wash, allow the pellet to air dry for 15 min.
   1. If the sample has not completely dried, air dry for another 15 min.

6. Rehydration of gDNA

1. Once the sample has dried, add 300 μl of Tris-EDTA to rehydrate the dried gDNA pellet.
2. Vortex the sample for 5 sec at medium speed and place in a 65 °C hot water bath for 1 hr.
3. Remove the samples from the water bath and incubate O/N at RT.
   NOTE: All products and reagents used are listed in the Materials Table, as well as Table 4.

Results

To determine optimal parameters for DNA extraction a series of paired DNA extractions was performed. A single saliva sample was split and each portion tested with one of two possible values for a given variable. At least eight replicates of each paired test were performed (e.g., a single saliva sample was aliquoted to test extraction both with and without initial 50 °C incubation). Optimization was based on four standard metrics: total DNA yield, the 260/280 value, the 260/230 value, and visual inspection of ele...

Discussion

The present procedure is an optimized DNA extraction protocol that has considerably improved yield of high molecular weight DNA compared to standard methods, without compromising DNA quality. The critical step with the biggest effect on yield the most was step 5.2, which includes a longer centrifugation step during ethanol precipitation than any published protocol reviewed here, except one that was not widely distributed¹¹. No changes in DNA quality associated with this longer centrifugation were detected, ind...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 ml Centrifuge Tubes	Fisher	12-565-268
Cell Lysis Solution	Qiagen	158908
Proteinase K	Sigma	P6556
Protein Precipitation Solution	Qiagen	158912
Isopropanol	Fisher	A416-4
Glycogen	EZ-BioResearch	S1003
70% Ethanol	Fisher	04-355-305
Tris-EDTA (TE)	Fisher	BP2473-1
NaCl	Fisher	AC194090010
Tris HCl	Fisher	BP1757-100
EDTA (0.5 M) Solution	Fisher	03-500-506
Sodium Dodecyl Sulfate	Fisher	BP166-100
Analog Vortex Mixer	Fisher	02-215-365
Centrifuge 5810R	Eppendorf	5811 000.010