Genomic DNA Isolation from Cheek Epithelium: A Step-by-Step Guide

Posted on Aug 27, 2024 in Chemistry

Genomic DNA Isolation from Cheek Epithelium

Exercise 1: Isolation of Genomic DNA from Cheek Epithelium Cells

Isolation and purification of nucleic acids is the first phase of the majority of procedures applied in molecular biology. Obtaining pure genetic material, regardless of its origin, is the basic aim of such work. Many methods exist for nucleic acid isolation and purification. Choosing the most suitable method depends on several factors:

  • The type of nucleic acid being analyzed (RNA/DNA)
  • The organism from which the genetic material is taken (plant, animal, bacterium)
  • The type of material from which the nucleic acid is isolated (tissue type)
  • The final purpose of the material (PCR, cloning, Real-Time PCR)

Biological materials from which DNA can be isolated include:

  • Blood
  • Smears (epithelium)
  • Sperm
  • Urine
  • Milk
  • Cerebrospinal fluid
  • Tissues
  • Bacterial and micro-organic cultures

Phases of DNA Isolation

1. Preparation of the Biological Material

Depending on the type and origin of the material, preliminary preparation may include cleaning off various external contaminations such as serum, culture medium, and remains of other cells. Pulverization and homogenization take place during this phase, followed by suspending a homogenous cell mass in a respective buffer and obtaining a pure sediment made only of the cells for DNA isolation.

2. Disintegration and Lysis of the Cells

Depending on the type of cells and tissues, cell disintegration may be carried out by different methods, for example:

  • Homogenization (soft animal tissues)
  • Detergent lysis (cultured cells)
  • Enzymatic lysis (bacterial and yeast cells)

When external membranes are damaged, DNA and other intracellular components are released. Therefore, it is important to maintain specific conditions so that the nucleic acid is not damaged. A salt solution is used (most often NaCl with Tris-HCl and EDTA) to dissolve DNA as well as for cell lysis. Proteinase K has a double function in DNA isolation: it is used for digestion and removal of protein contaminations, and it deactivates nucleases (enzymes that catalyze nucleic acid hydrolysis) that might degrade the nucleic acid during purification.

3. Separation of the Nucleic Acid from the Remaining Cell Components

Various types of salts and detergents are used for removing the ionic interaction between positively charged histones and other proteins, and the negatively charged DNA structure. Total deproteinization of the solution is performed by extraction with organic solvents, for example, a mixture of chloroform and alcohol.

4. Consolidation of the Isolated DNA

Upon extraction, nucleic acids are in the aqueous phase, contaminated by small-molecule compounds. By adding an organic solvent (e.g., ethanol or isopropanol), the polarity of the environment is decreased, which causes a decrease in the solubility of the DNA, which has an ionic structure. In this situation, DNA forms a thread-like sediment that may be collected by centrifugation. After precipitation and washing out with 70% ethanol, the DNA is left to dry out and then is suspended in a small amount of buffer of weak ionic strength (e.g., TE buffer) or in water. The isolated DNA may be stored at a temperature of 4-8°C for a month or at -20°C for a longer time (even a few years).

Presently, ready-made reagent kits are commonly used. They contain all the reagents ready for use in amounts suitable for DNA isolation.

Performing the Exercise

The aim of the exercise is to isolate genomic DNA from human cheek epithelium cells.

Notice! To avoid contamination of the isolated DNA, wipe the surface of the table with alcohol before starting the isolation, and wear nitrile gloves at all times!

I. Collection of the Material for the Tests

  1. Put on gloves!
  2. Take a sterile hygienic cotton swab out of its package.
  3. Swallow all saliva.
  4. Rub the inner side of the cheek forcefully with the cotton swab for nearly 1 minute. You may rub the cheek and gums. This will rub off the epithelium cells from which you will isolate DNA. The more you rub (and the less saliva rinses off the cells), the better the final result you will obtain. Do not eat food or smoke tobacco for about 30 minutes before collecting the material for the tests.

II. Isolation of the Genomic DNA from the Cheek Epithelium Cells

  1. After collecting the smear, detach a part of the cotton swab with the collected sample and place it in a tube.
  2. Add 0.7 ml of lysis solution RL and 20 μl of proteinase K to the tube.
  3. Mix thoroughly and incubate at 37°C for 20 minutes. Mix the sample from time to time by vortexing.
  4. Collect the entire solution from the tube and move it onto a column for DNA purification.
  5. Centrifuge the sample for 1 minute at 12,000 rpm.
  6. Move the column to a 2 ml tube and add 0.5 ml of A1 rinsing solution.
  7. Centrifuge for 1 minute at 12,000 rpm.
  8. Move the column to a new 2 ml tube and add 0.5 ml of A1 rinsing solution again.
  9. Centrifuge for 2 minutes at 12,000 rpm.
  10. Place the dried mini-column in a new 1.5 ml tube.
  11. Add 50 μl of elution buffer (10 mM TRIS.HCl, pH 8.5) previously heated to 75°C to the bottom of the mini-column.
  12. Incubate the sample for 3 minutes at room temperature.
  13. Centrifuge for 1 minute at 12,000 rpm.
  14. Remove the mini-column. The isolated DNA, ready for further analysis, will be at the bottom of the tube.
  15. Freeze the DNA at -20°C.

Materials and Equipment

  • DNA Isolation Kit for swab smears (A&A Biotechnology)
  • Eppendorf tubes
  • Automatic pipettes
  • Centrifuge (10,000–15,000 rpm)
  • Thermoblock
  • Vortex