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 sub....

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 so....

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