Saliva is becoming one of the more popular sample types for high-quality DNA extraction. In this article, we explore the merits of saliva as a source for DNA, the steps for extracting DNA from saliva as well as a few tips to ensure you get the best results. Let’s dig in.
The sample type you choose to extract DNA from depends on the requirements of your study and your study population.
While whole blood samples continue to be the “gold standard” in terms of the amount and quality of DNA you can extract, saliva is not far behind for a variety of reasons.
Why Choose Saliva for DNA Extraction?
The main reason many scientists are choosing saliva workflows to extract DNA from is saliva is an easy sample to collect.
It’s as easy as spitting into a collection tube. Compared to other methods like drawing blood and tissue biopsies, this method is entirely non-invasive and is more comfortable and convenient. This is especially important if your study cohort includes vulnerable populations. Or if you need to collect many samples throughout your study period, saliva can be the best way to ensure easy, consistent collection.
Beyond the ease of collection, saliva samples are also easy to store and transport. If you’re using a collection tube with an added stabilizer, saliva samples can remain stable for up to 5 years after collection at room temperature, depending on the collector. This means you don’t need to maintain a large freezer or spend extra on cold shipping.
Lastly, saliva offers sufficient DNA yields for most downstream applications. In many cases, it may not yield as much as large volume whole blood samples. However, saliva still yields more than enough pure gDNA for many processes, such as genotyping, whole-genome sequencing and other processes.
Steps for Extracting DNA from Saliva
Here is a brief overview of the steps involved for isolating pure gDNA from saliva. The volume of the sample and the throughput will depend on the collection tubes, instrumentation, and workflow you opt for, but they all generally follow these four steps.
Step #1: Cell Lysis
The first step to DNA isolation from any sample is cell lysis. This means breaking open the cells to release their DNA. In this step, cells are broken open and proteins are denatured through enzymatic digestion, causing DNA to be released.
Step #2: Protein Precipitation
As you may have noticed, cell lysis doesn’t just release DNA from the cell. It releases all types of cellular material. To separate the DNA from those other proteins, a purifier salt solution is then added to the saliva sample to concentrate and solidify proteins and lipids.
Step #3: Centrifugation
The solidified proteins and lipids are spun down and pelleted with centrifugation to separate from the rest of the solution.
Step #4: DNA Precipitation
The sample is then decanted into an isopropanol solution leaving behind the protein pellet. When the supernatant is added to the isopropanol this will concentrate and solidify the DNA to further remove any other impurities or contaminants.
Step #5: Centrifugation
The solidified DNA is then spun down into a pellet and separated from the rest of the solution.
Step #6: Washing
The DNA pellet is then washed with an alcohol solution to further clean the DNA pellet and remove any salt contaminants from the previous precipitation step.
Step #7: Re-suspension
A tris or water-based buffer is then added to the dried DNA pellet to bring it back into the solution, also known as resuspension or rehydration of the pellet. This resuspension is then usable in downstream applications. The buffer also helps to preserve the DNA in the case that it won’t be processed immediately after isolation.
Those are the basic steps used to isolate DNA from a saliva sample. If you want to ensure that your DNA yields are as pure and high in molecular weight as possible, here are a few additional tips you can follow.
How to Ensure the Best Results
Tip #1: Ensure Donors Follow the Instructions on the Tube
The first tip to ensure the best DNA yields from saliva collection is to make sure your study population and sample donors follow the instructions on the collection tube. To ensure you get the most DNA possible, they must fill the tube up to the line. Also, ensure they don’t eat, drink, smoke or chew gum at least 30 minutes before giving their sample.
Tip #2: Get Rid of the Bubbles
Make sure that there are no bubbles in the collection tube, if possible. Or, if there are bubbles in the tube, make sure they are above the required fill line. Bubbles are air and do not count towards the volume of saliva in a sample. Collecting less saliva means you collect less DNA.
Tip #3: Have a Plan for RNA
The resulting DNA pellet in many saliva sample workflows will still include RNA. In many cases, that’s not a problem, but if your downstream application will be affected by the presence of RNA, make sure to add a step to treat your samples with RNase to remove the RNA from the pellet.
Conclusion: Working with A DNA Extraction Expert Can Help
Optimizing your DNA yields from saliva can be a challenge. Following these steps and tips is a great way to start, but if you still need help narrowing down the right process or automating your saliva DNA extractions, get in touch with an expert like AutoGen. At AutoGen, our experts are always on hand to help you get the highest-quality yields from your saliva samples.