By Scott Rabuka on February 1, 20192019-02-01
Since the launch of this blog, The Genetic Link, in 2009, we have published numerous articles, interviews, technical information and infographics to share what we know about DNA from saliva. Saliva is the human body’s most accessible bio-fluid but, after 7 years, we continue to be surprised at how often we hear wrong perceptions about this robust and easy to access sample type. It’s time to set the record straight on DNA from saliva once and for all. Are you ready? Here are 8 facts about DNA from saliva that most people don’t know.
Fact # 1: DNA in saliva is derived from both buccal epithelial cells and white blood cells.
It might surprise you to know that much confusion surrounds the real source of genomic DNA in saliva. Surprisingly, most people assume the source of DNA in saliva is strictly buccal epithelial cells. However, studies show that up to 74% of the DNA in saliva comes from white blood cells which are an excellent source of large amounts of high quality genomic DNA. Yielding virtually the same amount of DNA per volume and the same DNA quality as blood, saliva can be considered equivalent to blood for genetic applications.
Fact # 2: The vast majority of DNA from saliva is of human origin.
While most scientists prefer large amounts of DNA, technological advances in downstream platforms now enable testing on even small amounts of DNA with the caveat that the DNA is of sufficient quality. As stated in fact # 1, the majority of DNA in saliva comes from white blood cells. However, human saliva also contains bacteria. When extracting DNA from saliva, bacterial DNA is recovered along with the human DNA. When compared to other oral sampling methods, such as buccal swabs or mouthwash, a 2mL saliva sample collected with Oragene yields approximately 11% bacterial DNA, substantially lower than mouthwash at 66% and cytobrushes at over 88% bacterial DNA.
If you are using saliva collected with a product like Oragene, you can be confident that the majority of DNA from saliva is of human origin with very low bacterial content.
Fact # 3: DNA from saliva is of high purity, despite low A260/A230 ratios.
Absorbance at 230 is used to measure various contaminants such as phenol and phenolic compounds, carbohydrates and other organics. Saliva samples contain a large amount of carbohydrates (from the heavily-glycosylated protein mucin). While protein is removed during extraction, small amounts of this carbohydrate is left behind. Carbohydrates absorb very strongly at 230nm so even small quantities of carbohydrate can greatly inflate the 230 reading leading to a poor ratio. The presence of these carbohydrates does not affect downstream application and therefore A260/A230 is not a useful method to assess the suitability for downstream use of DNA extracted from saliva samples. Phenolics can be of concern; however, these are not within Oragene and prepIT•L2P reagents, so this is not an issue for Oragene/saliva samples.
To accurately measure the purity of DNA extracted from saliva, A260/A280 should be calculated. The ratio of absorbance at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm. When extracting with prepIT•L2P the median A260/A280 ratio is between 1.6-1.9. These ratios are typically indicative of a DNA sample that will perform well on your downstream application given that all your other QC metrics pass (high molecular weight on gel, acceptable concentrations by fluorescent based quantification method).
Fact # 4: Saliva yields high quantity high-molecular weight DNA.
When extracting DNA from Oragene/saliva samples RNA will co-purify with DNA. The RNA will not affect your downstream applications (including PCR, SNP Genotyping, WES, or WGS) and can be removed, however it will affect quantitation of the DNA if quantifying only by absorbance. When quantifying by absorbance (such as Nanodrop) you are measuring the total amount of nucleic acids in a sample (RNA and DNA). Therefore, the amount of DNA in a sample could be overestimated and could result in reduced performance due to the under-loading of DNA into your assay. Quantification based on fluorescence such as Picogreen or Qubit provides an accurate measurement of DNA within a saliva sample.
The median yield of DNA from a 2 ml saliva sample using Oragene is 110 µg when purified according to the optimized Oragene protocol using prepIT•L2P and measured by the highly specific Fluorescence/DNase method. In terms of molecular weight, DNA from Oragene/saliva is >23 kbp in size. Both DNA yield and molecular weight will remain of high integrity for years at room temperature when collected in the Oragene stabilization chemistry.
Fact # 5: Saliva can reliably replace blood for DNA analysis.
Blood collection is often considered the golden standard for DNA quality and it is an established practice across hospitals, clinics, and labs worldwide. However, many people don’t know that replacing blood with saliva is a proven option for genomic DNA analysis.
Saliva collection kits (Oragene) are designed to stabilize high molecular weight DNA by inhibiting degradation and preventing bacterial growth. The majority of DNA obtained with Oragene is > 23kb in fragment size and the amount of bacteria has minimal practical significance as the vast majority is of human origin (average only 11.8% bacteria)3.
Multiple studies confirm DNA extracted from Oragene/saliva samples result in DNA of the highest integrity, performing equivalently to blood for the most demanding applications including microarrays and sequencing (targeted and whole genome).
Fact # 6: DNA from saliva is suitable for whole genome sequencing (WGS).
We are surprised that we continue to hear concerns about using saliva for sequencing and it is time to put those fears to rest once and for all. There are many scientific references for saliva (Oragene) being successfully used for sequencing in both small and large studies. For example, Dr. Cory McLean of 23andMe presented a poster in which he described WGS of 50 saliva samples. Dr. McLean’s work focused on the LRRK2 G2019S mutation in a Parkinson’s disease cohort, I encourage you to have a look at his poster. The DNA extracted from these archived Oragene/saliva samples was sequenced, using Illumina technology, to a median depth of 44.9 fold coverage and covered 97.8 – 98.2% of the genome. After identifying the variants in these samples Dr. McLean compared the results to data from the same cohort previously determined using a genotyping array and observed a 99.91 – 99.97% concordance, indicating that Oragene/saliva samples provide consistent results across different technology platforms. The question that many researchers continue to ask is: what impact does bacterial content from saliva have on sequencing? We’ve clearly demonstrated that when performing sequencing the bacterial content has no impact on variant calling.
In addition, a poster recently presented by the Broad Institute stated:
“To date, we have sequenced over 1585 (Oragene) saliva samples to 30x coverage using the HiSeqX (Illumina)… Given this experience, we are confident sequencing patient samples from (Oragene) saliva can be cost effective and produce high quality results for research and clinical studies.”
If you’ve been hesitant to use saliva for whole genome sequencing, you can be confident that it’s now time to give it a try.
Fact # 7: Collecting DNA from saliva is less expensive than DNA from blood.
The price associated with blood collection may be perceived to be free for many institutions that have established blood collection labs/service centres; however, there are real costs to sample collection even within these environments. Phlebotomists, medical supplies and shipping requirements (dry ice, containers, and overnight delivery) add an estimated $40 per sample, not including freezer storage. DNA from saliva, collected with a product like Oragene, in comparison, comes in a variety of formats with differing yield and stability capabilities which cost between 48% - 80% less. More savings are introduced as Oragene products enable at-home collection, standard shipping via regular mail at room temperature and zero refrigeration.
Daksis, J.I. et al. states, “The acquisition of high quality DNA for molecular assay from oral samples offers clear advantages in cost, handling, storing and shipping over acquisition of samples from blood. … It therefore opens the way for convenient point of care testing…”
Abraham J.E et al. continues “… commercial extraction of DNA from saliva is cheaper than from blood.”
And another study by Nishita, D.M. et al. reports “Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost.”
Overall, saliva is less expensive than blood for DNA collection when factoring in all costs associated with the sample collection procedure.
Fact # 8: DNA saliva sample collection methods are NOT all equal.
There are 3 methods for collecting oral DNA samples – dry, wet and non-invasive procedures. Dry procedures require the donor to insert a cytobrush, buccal swab or other collection device into the mouth where tissue is scraped from the gum and cheek surfaces. These methods collect primarily buccal cells and a high proportion of bacteria which stick to the gumline.
However, DNA samples collected from saliva where the donor spits into a collection device (Oragene) are quite different and offer higher yields and DNA quality than other oral DNA sample collection methods. One study, titled New Saliva DNA Collection Method Compared to Buccal Cell Collection Techniques for Epidemiological Studies, states:
“Whole-saliva collection provided an average DNA yield that was significantly greater than all other [oral] methods... Median yield [of Oragene/saliva]… was approximately three times the median yield of the oral rinse, and more than 12 times the median yields for the buccal swab and brush methods.”
It’s clear that NOT all DNA saliva methods are equal but you can be confident when choosing an option like Oragene which optimizes both quality and quantity of DNA.
These are the 8 facts about DNA in saliva that we believe many people don’t know. We hope this helps clear up much of the misinformation that continues to circulate about DNA from saliva. If you haven’t chosen DNA from saliva in the past, you should now feel confident in making that choice for any projects involving genomic DNA analysis. Tens of thousands of researchers and clinicians around the world are already working with saliva due to its high quality performance coupled with quick and easy collection and efficient transport. Be sure to request evaluation kits for your next study below.
 Thiede, C. et al. Buccal swabs but not mouthwash samples can be used to obtain pre-transplant DNA fingerprints from recipients of allogeneic bone marrow transplant. (2000). Bone Marrow Transplantation. 25(5): 575-577.
 Human genomic DNA content of saliva samples collected with the Oragene® self-collection kit, DNA Genotek white paper, PD-WP-011
 Iwasiow, R.M. and Birnboim, H.C. (2006). From turbidity to clarity: Simple methods to improve the A260/A280 ratio of Oragene®-purified DNA samples. DNA Genotek. MK-AN-017
 DNA yield with an Oragene® self-collection kit, DNA Genotek. PD-WP-001.
 Iwasiow, R.M. and Birnboim, H.C. (2011). Long-term stability of DNA from saliva samples stored in the Oragene® self-collection kit. DNA Genotek. PD-WP-005
 McLean et al, Whole-genome sequencing of 50 LRRK2 G2019S carriers discordant for Parkinson's disease,
 Dodge S, Ferriera S, Philippakis A, Farjoun Y, Banks E, Barry A, Wilkinson J, Cabili M, Sutherland S, Siedzik D, De Smet T, Gabriel S. Sequencing Whole Genomes with DNA Derived from Saliva. Poster session presented at: 2016 Advances in Genome Biology and Technology Meeting (AGBT); 2016 Feb 10-13; Orlando, FL.
 Ambrosone C.B. et al. Conducting Molecular Epidemiological Research in the Age of HIPAA: A Multi-Institutional Case-Control Study of Breast Cancer in African-American and European-American Women. J Oncol. 2009: 1-15 (2009).
 Daksis J.I. and Erikson G.H. Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples. PLoS ONE. 2(3): e305 (2007).
 Abraham, J.E. et al. (2012). Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping.
 Nishita D.M. et al. Clinical trial participant characteristics and saliva and DNA metrics. BMC Med Res Method. 9(71): 1-20 (2009).
 Rogers N.L. et al. New Saliva DNA Collection Method Compared to Buccal Cell Collection Techniques for Epidemiological Studies. Am J Hum Biol. 19:319–326 (2007).