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 Cells of Cheek Epithelium

Isolation and purification of nucleic acids is the first phase of the majority of procedures applied in molecular biology. Obtaining some pure material, regardless of its origin, is the basic aim of such works. There are many methods of nucleic acid isolation and purification, and a choice of the most suitable method depends on:

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

The biologic material that the DNA may be isolated from includes: blood, smears (epithelium), sperm, urine, milk, cerebrospinal fluid, tissues, bacterial and micro-organic cultures, etc.

Phases of DNA Isolation

1. Preparation of the Biologic Material

Depending on the type and origin of the material, preliminary preparation may include cleaning off various external contaminations, 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 pure sediment made only of the cell for DNA isolation.

2. Disintegration and Lysis of the Cells

Depending on the type of the cells and tissues, cell disintegration may be carried out by different methods e.g.: by 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 the cell lysis. Proteinase K is of double function in DNA isolation: it is used for digestion and removal of protein contaminations, as well as it deactivates nucleases (the enzymes catalyzing the nucleic acid hydrolysis) that might degrade the nucleic acid during their purification.

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

Various types of salts and detergents are used for removing the ion interaction between positively charged histones and other proteins, and negatively charged DNA structure. A total deproteinization of the solution is performed by extraction with organic solvents e.g.: 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 the organic solvent (e.g.: ethanol or isopropanol) the polarity of the environment is decreased, which causes a decrease in the solubility of the DNA that is of ion structure. In this situation, DNA forms thread-like sediment that may be collected by centrifugation. On precipitation and washing out with 70% ethanol, the DNA is left to dry out and then is suspended on a small amount of buffer of a weak ion strength (e.g.: TE buffer) or in water. The isolated DNA may be stored at the temperature of 4 – 8°C for a month or at -20°C for a longer time (even a few years).

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

Performing the Exercise

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

Notice! In order to avoid contamination of the isolated DNA, the surface of the table should be wiped with alcohol before the isolation is started, and nitrile gloves should be used in every case!

I. Collection of the Material for the Tests:

  1. Put the gloves on!
  2. Take a sterile hygienic cotton swab out of its package.
  3. Swallow the whole saliva.
  4. Rub forcefully the inner side of the cheek with the cotton swab for nearly 1 minute. You may rub cheek and gums. By doing so, you rub off the epithelium cells that you are to isolate DNA from. The more you rub (and the less saliva rinses off the cells), the better final result you are to obtain. No food should be eaten and no tobacco should be smoked for about 30 minutes before collecting the material for the tests.

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

  1. Collect 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 the whole and incubate at the temperature of 37°C for 20 minutes. Mix the sample from time to time by vortexing.
  4. Collect the whole solution from the probe and move 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 the temperature of 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 analyses, is to be found at the bottom of the tube.
  15. The DNA should be frozen at -20°C.

Materials and Equipment

  • – DNA Isolation Kit for swab smears (A&A Biotechnology)
  • – Eppendorf tubes
  • – automatic pipettes
  • – a centrifuge: 10,000 – 15,000 rpm
  • – a thermoblock
  • – a vortex