Introduction

Blood samples have traditionally been considered the gold standard DNA source for genomic analysis, but obtaining blood samples is a painful, invasive, and costly procedure that must be performed by qualified personnel.

By contrast, saliva collection is safe and non-invasive, and saliva collection kits can be mailed to donors for self-collection at home. Over the past two decades, saliva-based DNA collection has become routine for population-scale genetic studies, clinical research, and emerging diagnostic applications.

This article outlines how saliva is currently used for genetic studies, discusses technical considerations for sample processing and analysis, and explores future opportunities and challenges for this evolving field.

• Why use saliva as a source of genomic DNA?
• Genetic Analysis Techniques Used For Saliva-Based Studies
• The Challenges of Using Saliva DNA
• Genetic Research Using Saliva: Emerging Directions and Future Opportunities
• Conclusion
• GeneFix DNA/RNA Products

Why use saliva as a source of genomic DNA?

“Saliva is much easier and cheaper to collect and transport than blood.”

Recruitment and compliance rates are much higher when saliva samples are used instead of blood, as saliva collection is painless and can done at home. Study participants are often reluctant to provide blood samples due to the need to travel to a health centre or the painful nature of giving a sample. Another challenge when using blood samples is that they must be refrigerated or frozen during transport and storage.

Saliva samples do not require pre-processing and are commonly collected using non-hazardous reagents; therefore they can be sent to the laboratory via regular mail. Commercial saliva collection kits can stabilize DNA at room temperature for over five years, avoiding the high cost and logistical challenges associated with the cold chain transport of blood.

Saliva contains a mixture of buccal epithelial cells and leukocytes, from which high-quality genomic DNA can be extracted. In a study comparing DNA extracted from saliva and blood, Looi et al [i] (2012), the DNA yield from saliva of 7.8 µg/0.5 mL from a manual purification method was comparable to the DNA yield from blood using a salt precipitation method (7.4 µg/0.5 mL blood sample). DNA extracted from saliva and blood were both high purity (A260/280 > 1.70).

Genetic Analysis Techniques Used For Saliva-Based Studies

The range of genetic analysis tools used for saliva-based studies has expanded over the years. The key techniques are outlined below:

1. Single Nucleotide Polymorphism (SNP) Genotyping Arrays

SNP Genotyping arrays are microarray-based genotyping tools used for genome-wide or targeted genome screens. To run SNP microarrays, DNA samples of fragmented single-stranded DNA are hybridized to an array comprising hundreds of thousands of unique nucleotide probe sequences that target SNPs. SNP arrays can also be used to study copy number variations (CNVs), which are often associated with diseases.

SNP arrays are widely used platforms for direct-to-consumer applications such as ancestry analysis and large cohort population genetics studies. Arrays provide cost-effective, high-throughput analysis of hundreds of thousands of common SNPs, enabling genome-wide association studies (GWAS), ancestry inference, and polygenic risk score (PRS) calculation.

2. Next Generation Sequencing (NGS) Using Saliva Samples

NGS can provide data on thousands of genes at once from multiple samples, enabling the discovery and analysis of a wide variety of genome features. In general, NGS requires lower amounts of input DNA than microarrays, but the data analysis can be complex and costly. As costs come down and data analysis tools become more sophisticated, NGS is increasingly being used in clinical genetics, particularly for Mendelian disease diagnosis and pharmacogenomics.

NGS can be used to look at the whole genome or specific sections of interest. There are three main types of NGS:

Whole Genome Sequencing: As the name suggests, in Whole Genome Sequencing (WGS) the whole genome is sequenced, including non-coding regions, structural variants, and mitochondrial DNA. WGS is being adopted for research biobanks and clinical diagnostics as costs decline to extract as much data from samples as possible.

Targeted NGS: In Targeted NGS, samples are enriched for the areas of interest using hybridization capture or amplicon sequencing, and then only these areas are sequenced. Hybridization capture uses biotinylated oligonucleotide probes to capture the regions of interest, while amplification uses PCR. Targeted sequencing is faster and more cost-effective than WGS, allowing for deeper sequencing of areas of interest. Targeted sequencing is an especially sensitive and powerful method for identifying variants and mutations, including rare variants.  

Whole Exome Sequencing (WES): Only the protein-coding regions (~1–2% of the genome) are sequenced in WES, often including rare or clinically significant variants. Traditionally whole exome sequencing was performed instead of WGS, to keep costs down.

3. Mitochondrial DNA Analysis

The mitochondrial genome mutates much faster than the nuclear genome, making mitochondrial DNA a valuable tool for systematics, evolutionary biology research, population genetics, and conservation biology research.

Mitochondrial DNA (mtDNA) can also be used for genetic epidemiological studies, and forensics.

4. PCR

Saliva DNA can be amplified using PCR for genotyping or diagnostic applications. Ng et al[ii] (2006) studied the performance on saliva DNA samples in real time PCR and found that saliva was a viable alternative source of human genomic DNA for genetic epidemiological studies.

The use of saliva as a source of DNA for PCR-based diagnostic tests came to the fore during the COVID-19 pandemic, and a study by Ganie et al (2023)[iii] investigated the sensitivity and specificity of saliva as a non-invasively-obtained specimen for molecular detection of SARS-CoV-2 RNA.

5. DNA Methylation Studies

In 2019, Murata et al[iv], found that in addition to high correlation in DNA methylation profiles, CpG sites showing large interindividual DNA methylation differences were similar between blood and saliva, so saliva could be an alternative source of genomic DNA for cohort studies, as long as source‐dependent DNA methylation differences were considered.

The Challenges of Using Saliva DNA

Using saliva for genetic analysis presents several technical and operational challenges. However, in recent years, there have been significant improvements in devices to collect saliva and good yields of high-quality DNA can now be extracted.

• Variable DNA Quality: Degradation due to improper storage or low epithelial cell content can affect downstream applications, especially WES and WGS. DNA integrity is crucial for detecting structural variants or phasing haplotypes.
• Microbial Contamination: Co-extraction of bacterial DNA can reduce sequencing efficiency. Metagenomic filtering and human-specific capture protocols help mitigate this, though they increase cost and complexity.
• Sample Identity and Mix-ups: Self-collection increases the risk of sample mix-up or contamination. Barcoding, digital tracking, and sample fingerprinting via SNP profiles are used to verify identity and minimize these issues.

Genetic Research Using Saliva: Emerging Directions and Future Opportunities

1. Liquid Biopsy Applications

Saliva is being explored as a non-invasive liquid biopsy medium, particularly for cancers localized to the oral cavity, pharynx, or lungs. ctDNA and exosomes can be isolated from saliva and analyzed via droplet digital PCR (ddPCR), targeted sequencing, or methylation assays. While sensitivity remains lower than plasma-based liquid biopsies, saliva offers a complementary diagnostic approach.

2. Pharmacogenomics and Precision Medicine

Clinical-grade saliva tests are being developed to guide therapeutic decisions based on variants in genes such as CYP2D6, SLCO1B1, and DPYD. Saliva-based assays offer a valuable route for predictive testing in primary care, psychiatry, and oncology.

3. Decentralized Clinical Trials and Global Health

Saliva-based genotyping supports remote enrolment and sample collection in decentralized clinical trials. This is especially valuable in rare disease research and studies requiring diverse representation. With minimal infrastructure needs, saliva testing can enhance genetic screening in low- and middle-income countries.

4. Genetic Epidemiology and Behavioural Studies

Saliva-based DNA collection has facilitated the expansion of socio-genomic research, including studies on educational attainment, cognitive function, and mental health disorders. Twin studies and family-based designs increasingly rely on at-home kits for sample collection, improving retention and longitudinal follow-up.

5. AI and Predictive Modelling

The growing volume of genotypic and phenotypic data from saliva-based studies is well suited to machine learning approaches. AI models are being used to improve variant interpretation, identify complex trait associations, and predict treatment outcomes from polygenic and environmental features.

6. Multi-omic Profiling

Saliva contains not only DNA but also RNA, proteins, metabolites, and microbial communities. Recent studies have demonstrated the feasibility of salivary transcriptomics, proteomics, and microbiome sequencing, paving the way for integrated multi-omic analyses.

These data types could enable early disease detection, especially in inflammatory, metabolic, or neurodegenerative conditions. For example, salivary miRNAs and extracellular vesicles are being investigated as biomarkers for Alzheimer’s and head & neck cancers.

Conclusion

Saliva has emerged as a practical, scalable biospecimen for genomic research and clinical applications. It enables high-throughput sample collection, democratizes access to genetic testing, and supports multi-omic innovation. While challenges in sample quality, microbial contamination, and data interpretation persist, ongoing technical advances are steadily improving the reliability and breadth of saliva-based assays.

As we move toward a more personalized, preventative healthcare model, saliva will likely serve as a cornerstone of population genomics and precision medicine—offering a low-friction path from collection to insight.

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Related Blogs:

• To read more about using saliva instead of blood for genetic testing, click here: https://isohelix.com/saliva-instead-of-blood/
• For a discussion of the DNA extraction methods for saliva click here: https://isohelix.com/the-best-dna-extraction-methods-from-saliva-or-buccal-swabs/

[i] Looi ML, Zakaria H, Osman J, Jamal R. Quantity and quality assessment of DNA extracted from saliva and blood. Clin Lab. 2012;58(3-4):307-12. PMID: 22582505

[ii] Ng DP, Koh D, Choo S, Chia KS. Saliva as a viable alternative source of human genomic DNA in genetic epidemiology. Clin Chim Acta. 2006 May;367(1-2):81-5. doi: 10.1016/j.cca.2005.11.024. Epub 2006 Jan 4. PMID: 16388788.

[iii] Ganie, M. W., Nainggolan, I. R. A., Bestari, R., Hazidar, A. H., Hasibuan, M., Siregar, J., Ichwan, M., Kusumawati, R. L., & Lubis, I. N. D. (2023). Use of saliva as an alternative diagnostic method for diagnosis of COVID-19. IJID Regions, 8, S8-S12. https://doi.org/10.1016/j.ijregi.2023.03.011

[iv] Murata Y, Fujii A, Kanata S, et al. Evaluation of the usefulness of saliva for DNA methylation analysis in cohort studies. Neuropsychopharmacol Rep. 2019;39(4):301-305. doi:10.1002/npr2.12075