Chapter 9 :DNA Extraction From Saliva

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Saliva is a readily available and non-invasive source of DNA, making it valuable for various genetic analyses and diagnostic tests. One common method for extracting DNA from saliva samples is using a silica column. This blog post will guide you through the general steps involved in this process.

Materials Required

  • Saliva sample
  • Lysis buffer
  • Proteinase K
  • RNase A
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Silica columns
  • Collection tubes
  • Microcentrifuge

Procedure

Sample Preparation and Lysis:

  1. Collect a saliva sample using a collection kit or by spitting directly into a sterile container.
  2. Add lysis buffer to the saliva sample. This buffer contains detergents and enzymes, such as Proteinase K, that break down cell membranes and proteins, releasing DNA into the solution.
  3. Incubate the sample at an appropriate temperature to ensure complete lysis.

RNA Removal:

Add RNase A to the lysate to degrade RNA, which can interfere with downstream applications.

DNA Binding to Silica Column:

  1. Add binding buffer to the lysate. This buffer creates conditions that promote DNA binding to the silica membrane within the column.
  2. Transfer the lysate to a silica column.
  3. Centrifuge the column to bind DNA to the membrane and remove impurities.

Washing:

  1. Wash the silica column with wash buffer to remove any remaining contaminants.
  2. Centrifuge the column to remove the wash buffer.

DNA Elution:

  1. Add elution buffer (low salt buffer or water) to the silica column.
  2. Centrifuge the column to elute the purified DNA into a collection tube.

Tips and Considerations

  • Follow the instructions provided with the specific saliva collection kit or protocol you are using.
  • Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
  • Optimize lysis and binding conditions for efficient DNA extraction.
  • Handle saliva samples with care and follow appropriate safety guidelines.

Applications of Extracted Saliva DNA

The DNA extracted from saliva using the silica column method can be used for various applications, including:

  • Genetic testing and screening
  • Ancestry analysis
  • Forensic investigations
  • Research studies

Conclusion

Silica column-based DNA extraction from saliva is a simple and effective method for obtaining DNA for various applications. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality DNA from saliva samples.

Lysis Buffer :

DNA lysis buffer is a solution that breaks open blood cells to extract DNA. It’s used in molecular biology experiments to analyze DNA.

Binding Buffer :

A binding buffer is a reagent used to bind DNA to silica during DNA extraction. It helps to purify DNA from various samples, including blood, cells, and tissues. It contains Guanidine Hydrochloride and Nuclease free water

Washing buffer :

DNA Wash Buffer (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)

Elution buffer :

(0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )

Saliva sample collection steps:

1. Do not eat, chew gum, drink or smoke for 30 minutes prior to saliva collection.

2. Rinse your mouth with water and wait for 10 minutes. 

3. Spit saliva into a 5ml collection tube up to the 2ml scale position. (Do not spit the sputum into the collection tube. If the saliva is not enough, you can do the tongue movement to promote secretion. A small amount of foam floating on the upper layer of the saliva does not include calculation in the 2ml saliva collection; the collection process must be completed within 30 minutes)


Saliva DNA Extraction Protocol

  • Take 1 ml of Saliva sample and transfer it to sterile microfuge tube.
  • Add 200μl of Lysis buffer + 20μl Proteinase K +10 μl Rnase Aand Mix thoroughly by vortexing.
  • Incubate at 65°C for 15-20 minutes either on thermo mixer or water bath or dry bath. Briefly invert the tube once during incubation.
  • After incubation add 200μl Isopropanol to the lysate. Mixed by inverting a tube 15- 20 times.
  • Transfer entire lysate to the DNA spin column, and centrifuge at 12,000 rpm for 2 mins. Discard the flow through liquid 
  • Repeat above step, until the entire sample has been processed and retain column for further processing. 
  • Place the column into the same collection tube. Add 500μl of Binding buffer. Centrifuge at 12,000 rpm for 2 min. discard the flow through.
  • Place the column into same collection tube. Add 500μl of Wash buffer. Centrifuge at 12,000 rpm for 1 min. discard the flow through.
  • Place empty DNA spin column, with the lid open into the same collection tube and centrifuge at 12,000 rpm for 2 min.
  • Place the column into a new sterile 1.5 ml eppendorf tube, add 30 μl preheated Elution buffer. Incubate at room temperature for 5 min. (perform this step twice 30 μl + 30 μl = 60 μl)
  • Centrifuge 10,000 rpm for 1 min to elute pure blood gDNA. The first elution normally yields 60-70 % of DNA bound. A second elution with another 30 μl buffer will yield another 20 % of the DNA.
  • Note: Elution volume may vary as per downstream process
  • Discard the Column, and save elute. Do not reuse binding columns or collection tubes.

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