To carry out any genetic analysis or study, the adequate collection of DNA is a critical and fundamental step. As opposed to blood as a traditional source of genomic DNA , alternative methods have emerged that allow the extraction and extraction of DNA from saliva , with notable advantages, including:
- High quantity and quality of DNA obtained
- Non-invasive or painful method for the patient
- DNA stability at room temperature for years
In this post we solve some of the most frequently asked questions when it comes to obtaining, stabilizing and extracting DNA from saliva with the Oragene · DNA kits.
– COLLECTION OF DNA FROM SALIVA –
- What DNA performance can be obtained in saliva samples?
The yield of DNA from saliva samples varies depending on the kit with which they are collected, and can reach up to 110ug of DNA of medium yield.
Here you will find more information about the characteristics of the different collection kits, and the performance that is obtained with each of them.
- What is the best method to quantify that DNA performance?
Quantification with a fluorescent marker such as SYBR Green or PicoGreen is recommended as these markers bind to double-stranded DNA.
Absorbance quantification may overestimate the amount of DNA due to the sum of the absorbance of the RNA co-purified with the DNA of interest.
- Why can you get a lower average DNA yield?
This may be due to multiple factors, including:
– The DNA present in the saliva always has a degree of variability (even in the same donor, the amount of DNA present in the saliva can vary from day to day).
– The DNA yield may vary depending on the purification method used.
- What can be done to increase DNA performance?
To optimize DNA performance it is very important to collect the volume of saliva indicated in each kit.
- What is the source of DNA in the saliva?
The DNA in the saliva comes from the white blood cells and the buccal epithelial cells.
- How much bacterial DNA does the saliva sample contain?
The amount of bacterial DNA depends on the method with which the samples are collected:
– The samples collected with the Oragene · DNA kits contain <12% of bacterial DNA .
– Mouthwash samples have approximately 50% bacterial DNA.
– Samples taken using traditional swabs contain almost 90% of bacterial DNA.
- How can saliva samples be collected in children and / or in patients with spitting difficulties?
– SAMPLE STORAGE –
- At what temperature should the saliva samples be stored?
Samples collected with Oragene · DNA kits are stable at room temperature for years. For longer term storage samples can also be frozen at -20ºC or -80ºC.
Refrigeration at 4ºC is not recommended.
- Can samples withstand freeze-thaw cycles?
Yes, samples collected with Oragene · DNA can withstand at least 5 freeze-thaw cycles without evidence of DNA degradation.
– DNA PURIFICATION FROM SALIVA –
- What can be done to improve the A260 / 280 ratio of purified DNA?
Some of the tips include:
– Make sure that the samples have been incubated for at least one hour at 50ºC before purification.
– Make sure not to alter the protein pellet after the first centrifugation step.
– Make sure that the spectrophotometer is subtracting the A320 values from the A260 and A280 values.
– Make sure that the values of A260 and A280 are in the range of 0.1 to 1.0.
- By what methods can DNA be purified from saliva samples?
DNA from saliva can be purified by:
– Ethanol precipitation
– Phenol / chloroform extraction
– Commercial kits such as QIAmp (Qiagen) or PUREGENE (Gentra)
- Is it possible to automate purification using DNA extraction robots?
Yes, BioRobot EZ1 (Qiagen), AutoPure (Gentra), Magtration 12GC (PSS Bio), Tecan or Biomek NX / FX can be used, among others.
- When running an agarose gel, does the stain at the bottom correspond to degraded DNA?
No, that stain corresponds to low molecular weight RNA.
- How can RNA be removed from saliva samples?
We leave you this protocol to remove RNA from saliva samples.
– APPLICATIONS –
- What applications are compatible with the DNA obtained from saliva samples?
DNA from saliva is compatible with most applications such as PCR, real-time PCR, multiplex PCR, SNP genotyping, microarrays, sequencing, RFLP, etc.
- Can complete genome amplification (WGA) be performed from saliva DNA?
Yes, DNA from saliva can be used with commercial WGA kits such as GenimiPhi (GE Healthcare), GenomePlex (Sigma-Aldrich), and REPLI-g (Qiagen).
- Can the bacterial DNA present in the sample interfere with the application?
The DNA from saliva collected with the Oragene · DNA kits works exactly the same as DNA obtained from blood samples for any application, since the amount of DNA present in the sample is minimal.