Exercise 4: DNA Isolation from Oral Cavity Epithelium - PDF

WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise 4: Isolation of genomic DNA from oral cavity epithelium and analysis of its purity Theory Student:  Knows DNA composition,  Knows and understands the goals and methods of DNA isolation  C...

WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise 4: Isolation of genomic DNA from oral cavity epithelium and analysis of its purity Theory Student:  Knows DNA composition,  Knows and understands the goals and methods of DNA isolation  Can evaluate the quality of isolates based on spectrophotometric measurements. Structure of DNA Deoxyribonucleic acid (DNA) is found, among other places, in the cell nucleus, where it forms chromosomes. It serves as the carrier of genetic information for living organisms and sometimes viruses. DNA is an unbranched, linear biopolymer, with its monomers being nucleotides. Nucleotides are composed of one of four nitrogenous bases: adenine, guanine, cytosine, or thymine, which are connected to a five-carbon sugar, deoxyribose. The first carbon atom of deoxyribose is linked to the base through an N-glycosidic bond. Additionally, the hydroxyl groups at the third and fifth carbon atoms of deoxyribose are linked (esterified) to the phosphate residue. In this way, successive nucleotides in the chain are connected to each other, forming what are known as 3',5'-phosphodiester bonds (Fig. 1). Adenine and guanine are referred to as purines and are bicyclic compounds. Cytosine and thymine are pyrimidines - monocyclic compounds. DNA typically exists in the form of two strands (double-stranded DNA), running in an antiparallel manner, meaning one end of one strand is precisely opposite the beginning of the other strand, creating a right-handed double helix winding around a common axis. The nitrogenous bases are oriented towards the interior and form base pairs, while the sugar and phosphate residues, connected to each other by a phosphodiester bond, are on the outside of the helix. So, guanine always pairs with cytosine, and thymine pairs with adenine. Hydrogen bonds form between the bases, stabilizing the helical structure. Three hydrogen bonds form between G-C pairs, while A-T pairs have two hydrogen bonds. Therefore, G-C bonds are stronger than A-T bonds. The way the bases of the two DNA strands pair up is called complementary base pairing. The sequence of one DNA strand is complementary to the sequence of the other. This allows one strand to be used for replication or to repair the other strand in case of damage (Fig. 1). Page 1 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Fig. 1. Structure of DNA. Creative Commons Attribution-Share Alike 2.5 Generic. DNA Research Many scientific works by researchers, medical biologists, or diagnosticians require the initial isolation of DNA from various research or clinical materials. The isolated DNA finds application in: detecting genetic diseases, assessing predisposition to the development of tumors, producing next-generation drugs, cell therapy, genetic engineering. The goal of DNA isolation is to obtain a high-molecular-weight preparation, purified from proteins and DNA enzyme inhibitors, with maximum efficiency. The choice of the appropriate isolation method depends on: the type of nucleic acid you want to obtain (genomic DNA, mitochondrial, plasmid), the origin (plant, animal, bacterial, viral, etc.), the type of material from which the isolation is carried out (tissues, organs, cell culture, etc.), Page 2 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 the expected results (purity, quality, isolation time, etc.), the purpose (PCR, cloning, etc.) For molecular studies of humans, DNA is most commonly extracted from peripheral blood. Blood should be collected in an EDTA (ethylenediaminetetraacetic acid) tube, which prevents blood clotting and inhibits the activity of deoxyribonucleases. In human studies, DNA is also isolated from: epithelial cells, fibroblast cultures, amniotic fluid cells (AFC), chorionic villi (CVS), hair follicles. Less commonly used materials for diagnostic studies include: blood spots, semen, tissue fragments obtained by fine needle biopsy, and bone marrow. Stages of DNA Isolation Regardless of the procedure used, most methods consist of several fundamental stages of isolation: Stage 1. Preliminary preparation of the biological material for DNA isolation, including purification, fragmentation, homogenization, and suspension in a buffer. Stage 2. Cell disintegration and lysis, releasing DNA into a solution where it is soluble and protected from degradation. Stage 3. Purification, separating the nucleic acid from other cellular components. Stage 4. Concentration of the DNA preparation and removal of low-molecular-weight impurities. Stage 5. Qualitative and quantitative evaluation of the isolated DNA. Determination of the Purity and Concentration of DNA and RNA Preparations Purine and pyrimidine rings in nucleic acids absorb UV light. DNA containing all nitrogenous bases exhibits maximum absorbance at a wavelength of 260 nm, which is the averaged value of the individual nitrogenous base wavelengths absorbances. Protein exhibits maximum absorbance at a wavelength of 280 nm, and contaminants like organic compounds absorb at 230-240 nm. Page 3 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Assessing the purity and concentration of a DNA solution is based on measuring light absorption (optical density, OD) at wavelengths of 260 nm (maximum absorption for DNA and RNA) and 280 nm (maximum absorption for proteins) using a spectrophotometer. The OD260/OD280 ratio (A260/A280) describes the purity of a DNA or RNA preparation. Pure double-stranded DNA (dsDNA) should have an A260/A280 ratio of approximately 1.8. For pure RNA, the A260/A280 ratio should be around 2.0. A value below 1.8 indicates the presence of proteins in the preparation. An A260/A280 ratio of 1.5 indicates protein contamination of around 50%. The spectrophotometric measurement also allows us to make a quantitative assessment of DNA. To calculate the concentration of DNA, the following formula can be used: 𝐶 ∗𝐴 𝐶 = 𝐴 Cb= concentration of the analyzed sample, Ab= absorbance of the analyzed sample, Cst= concentration of the standard, Ast= absorbance of the standard. Isolated DNA can be stored for a short time at +4 C, but ideally at -20 C. DNA Isolation Methods Solvent-based methods: DNA isolation using phenol and chloroform extraction (organic method). Chromatographic methods: DNA isolation by binding DNA to a carrier (silica columns), DNA isolation using magnetic separation. DNA Isolation using Phenol and Chloroform Extraction To degrade cell membranes and denature proteins, a homogenization process of the tested material is carried out using detergents [sodium dodecyl sulfate (SDS), Triton-X100]. Cell lysis is performed at an elevated temperature in the presence of proteinase K (an enzyme that degrade proteins). The obtained lysate is then vigorously shaken several times in the presence of phenol and chloroform, which extract lipids from the aqueous phase (the obtained lysate). The isolated sample can be divided into the following layers: Upper layer - aqueous, containing nucleic acids, Middle layer - containing denatured proteins, Page 4 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Lower layer - organic. DNA precipitation occurs in the presence of sodium ions using isopropanol or ethanol. The resulting DNA pellet, after ethanol washing, is dissolved in water or TE buffer (Tris-HCl). Column-based DNA Isolation During DNA isolation, the sample undergoes lysis in the presence of buffers that stimulate the breakdown of tissue and cell structures and release DNA. The sample solubilization (increasing solubility) is assisted by proteolysis. Proteinase K deactivates DNA-digesting enzymes, known as DNases. It digests proteins, including DNA-binding proteins, and breaks down cellular debris, improving the isolation efficiency. The proteinase is activated at an elevated temperature during the first stage of isolation. Subsequently, buffers and ethanol are added. These compounds create conditions for the selective binding of DNA to the GeneMATRIX column. Then, during a brief centrifugation, DNA binds to the column, while the unbound impurities remain in the filtrate from the column. Their trace residues on the column are effectively removed during two washing steps. Elution of the purified DNA is carried out using a low-salt buffer, e.g., containing Tris-HCl, TE, or distilled water. The purified DNA preparation is suitable for direct use and does not require further ethanol precipitation. DNA Isolation using Magnetic Method The procedure begins with cell lysis in the presence of proteinase K and a lysis buffer. Then, a suspension containing magnetic beads is added, which bind DNA molecules. The beads with bound material are exposed to a magnet, immobilizing them. Nucleic acids bound to the beads are washed several times to remove impurities. Subsequently, the magnet is removed, and the DNA bound to the beads is recovered using an appropriate elution buffer. The mixture is centrifuged to remove the beads. Texts required for independent reading by the student prior to class (required for test): Bruce Alberts, Dennis Bray, Karen Hopkin: 'Essential Cell Biology, Volume 1', Scientific Publisher PWN. Warsaw 2019. Chapter 5 DNA and chromosomes, pages 174-198 (available via ibuk). Page 5 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Practical Part Student:  performs a swab from the inner surface of the oral cavity epithelium,  isolates DNA from the swab,  evaluates the quality and purity of the isolate. Isolation of DNA from epithelial cells collected from the inner cheek surface Equipment: GeneMATRIX Swab-Extract DNA Purification Kit, 1.5 ml and 2ml DNase-free tubes, Ethanol [96-100%], DNase-free pipettes and pipette tips, Vortex, Laboratory centrifuge, Thermal block, Spectrophotometer. Before commencing the DNA isolation procedure from epithelial cells, activate the mini- column by completely saturating the membranes with an activation buffer. To do this, add 30 µl of Buffer S activation buffer to the mini-column (do not centrifuge) and then leave it at room temperature until applying the lysate to the mini-column. 1. Procedure for collecting epithelial cells from the inner cheek surface using a swab stick. CAUTION For efficient isolation of cheek epithelial cells, avoid drinking, eating, smoking, or brushing teeth approximately 1-2 hours before isolation. There will be time allocated for breakfast during the session. Page 6 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Fig. 2. Sterile swab stick by COPAN 1. Open the package by twisting and then removing the red cap. 2. Take out the swab stick with the cotton tip from the tube, being careful not to touch the cotton with your fingers. 3. Place the cotton tip in your mouth and rub firmly against the inner cheeks while holding the cheek on the outside with your fingers. To obtain an optimal number of cells, rub each cheek for approximately 30 - 40 seconds using a different side of the cotton tip for each cheek. Do not press too hard to avoid breaking the stick or detaching the cotton. 4. Insert the cotton tip into the tube (2ml with a rounded bottom), snap off the stick at the marked point, and close the tube with the cap. 2. The procedure for isolating DNA from cheek epithelial cells 1. Add 400 μl of Lyse S buffer and 10 μl of Proteinase K. 2. Mix by inverting the tube several times or vortexing and incubate for 30 min at 56°C. Mix by inverting every 10 min. 3. Add 400 μl of Sol S buffer and mix thoroughly by inverting the tube several times. 4. Incubate for 10 min at 70°C. 5. Add 200 μl of ethanol (96–100%). Mix thoroughly by inverting the tube several times. 6. Centrifuge the tube with the swab stick for 2 min at 11 000 x g. 7. Transfer 600 μl of the lysate to the DNA binding spin-column and centrifuge at 11 000 x g for 1 min. 8. Remove the spin-column, pour off supernatant and place back into the receiver tube.  Continue centrifugation, if not all of the lysate passed through the column. 9. Transfer 300 μl of the lysate to the same DNA binding spin-column and centrifuge at 11 000 x g for 1 min. 10. Remove the spin-column, pour off supernatant and place back into the receiver tube. 11. Add 500 μl Wash SX1 buffer to the spin-column and centrifuge for 1 min at 11 000 x g. 12. Take out the spin-column, discard flow-through and place back the spin-column in the collection tube. Page 7 of 8  WORKSHEET 4- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 13. Add 500 μl Wash SX2 buffer to the spin-column and centrifuge for 2 min at 11 000 x g. 14. Place the spin-column in a new collection tube (1.5 ml) and add 50 μl of Elution buffer to elute bound DNA.  To avoid transfering traces of DNA between the spin-columns do not touch the spin-column walls with the micropippete. 17. Incubate the spin-column/collection tube assembly for 2 min at room temperature. 18. Centrifuge the spin-column for 1 min at 11 000 x g. 19. Discard spin-column, cap the collection tube. DNA is ready for analysis/manipulation. It can be stored either at 2–8°C or at -20°C. 3. Quantification and quality assessment of the isolate using a spectrophotometer. The expected yield of isolation is 1 to 10µg DNA (10 to 100ng/µl). Record the results for the sample! Group, sample initials:…………………... Concentration of DNA ng/µl:…………. Purity for the ratio 260/280nm=……………. Purity for the ratio 230/260nm=……………. Page 8 of 8