What is the purpose of extracting DNA from cells?

Extracting DNA from cells has multiple purposes in research, diagnostics, and industry, such as genetic analysis, which enables the classification of species through techniques like 16S rRNA sequencing and whole-genome sequencing. It is also fundamental in molecular cloning and genetic engineering, where bacterial DNA is manipulated to create recombinant DNA and express proteins. DNA extraction also allows the identification of antibiotic resistance genes, helping to track and combat the spread of resistant strains. As a diagnostic tool, bacterial DNA is essential for detecting pathogens through assays such as PCR (polymerase chain reaction), which are used in clinical, food safety, and environmental monitoring. The extraction of DNA is also a critical first step in metagenomics, enabling the study of microbial communities and their functions in various ecosystems. Additionally, bacterial DNA is used to study gene function and regulation, providing insights into bacterial physiology, metabolism, and adaptation. In biotechnology and synthetic biology, DNA extraction underpins the engineering of bacteria for applications such as biosensors, biofuels, and the production of valuable biomolecules. It also facilitates evolutionary and comparative genomics studies, shedding light on genetic variation, evolution, and horizontal gene transfer among bacteria. DNA extraction plays a role in vaccine development by identifying virulence factors and antigens, which can be targeted for preventing bacterial infections. In forensic microbiology, extracted DNA helps trace contamination sources and analyze microbial evidence. Finally, it is crucial in probiotic and fermentation research, allowing for the identification and optimization of beneficial bacterial strains used in health and industrial applications.

What are the steps, reagents and materials to extract DNA?

DNA extraction from cells involves isolating and purifying DNA from various types of tissues or cells. The process generally includes two main steps: (1) disruption of the cell membrane (and the cell wall, if present, as in plant cells) to release the DNA, and (2) purification, which involves separating the DNA from other cellular components and debris. First, the cell membrane is disrupted using a combination of the methods described in table 1 to get a fluid containing all the cell components, including the DNA. This process is called cell lysis and the resulted fluid is known as lysate (Figure 1).

Figure 1. Cell lysis and lysate.

Table 1. Methods for cell disruption

During cell lysis, various chemicals and reagents are used to break down different cellular components. Detergents and surfactants disrupt lipids, proteinases degrade proteins, and RNA is either enzymatically degraded or selectively removed through binding agents. Next, a concentrated salt solution is added to the lysate to aggregate the disrupted cell components, leaving the DNA dissolved in the solution. The lysate is then centrifuged at high speed or filtered through membranes to separate the DNA from the cellular debris. Finally, the DNA is precipitated, concentrated or bound to resins (Figure 2).

Figure 2. Reagent-based and column-based DNA purification approaches.

Composition and function of the different reagents commonly used for DNA extraction

• Tris/EDTA (TE Buffer) 
  • Act as a buffering agent to maintain a stable pH (~ 8.0), which protects DNA from degradation during extraction.
• EDTA (Ethylenediamine-tetraacetic acid)
  • Functions as a chelating agent that binds divalent metal ions like Mg²⁺ and Ca²⁺, essential cofactors for DNases (DNA-degrading enzymes).
  • Inhibits DNase activity by binding these ions, protecting DNA from degradation.
• NaCl/KCl (Sodium Chloride/Potassium Chloride)
  • Stabilize DNA by neutralizing the negative charges on the DNA phosphate backbone, reducing repulsion between DNA strands.
  • Promote DNA precipitation during extraction and assist in removing proteins and contaminants by altering their solubility.
• Detergents (e.g., SDS, Triton X-100)
  • Disrupt cell membranes by solubilizing lipids and proteins, lysing cells and releasing contents, including DNA.
  • Denature proteins to ease their removal during purification.
• Reducing Agents (e.g., DTT, β-Mercaptoethanol)
  • Break disulfide bonds in proteins, aiding in their denaturation and inactivating enzymes like DNase, which could degrade DNA.
• Enzymes (e.g., Proteinase K, RNase, Lysozyme)
  • Proteinase K digests proteins, including those bound to DNA, aiding in their removal.
  • RNase degrades RNA, ensuring RNA-free extracted material.
  • Lysozyme breaks down bacterial cell walls, especially in gram-positive bacteria, to access DNA.

What are the steps to purify DNA from the lysate?

Purification, the separation of  the DNA from the rest of the lysate components, can be accomplished by three different methods:

• Ethanol Precipitation

Ethanol precipitation is a widely used method to purify and concentrate DNA from aqueous solutions. In this process, DNA is precipitated by adding ethanol (or isopropanol) and a salt, such as sodium acetate, which neutralizes the charges on the DNA phosphate backbone, making it less soluble in water. The addition of cold ethanol causes the DNA to precipitate out of the solution, allowing it to be collected by centrifugation. This method is effective at removing small impurities, salts, and solvents from the DNA. It is cost-effective and suitable for precipitating DNA from large volumes, however it may co-precipitate some contaminants, such as proteins or polysaccharides, and requires careful handling to avoid DNA loss.

• Phenol/Chloroform Extraction

Phenol/chloroform extraction is used to separate DNA from proteins and other cellular debris based on their solubility in organic solvents. The lysate is mixed with phenol and chloroform, creating a biphasic mixture. DNA remains in the aqueous phase, while proteins and lipids partition into the organic phase or interphase. After centrifugation, the aqueous phase containing the DNA is carefully collected, leaving behind impurities. This method is highly effective at removing proteins and other contaminants. It produces high-purity DNA with minimal protein contamination, but it uses toxic and hazardous chemicals that require careful handling and proper disposal, and it can be time-consuming.

• Column Purification

Column purification uses silica-based columns that selectively bind DNA in the presence of high salt concentrations. The lysate is passed through the column, and DNA binds to the silica membrane while contaminants such as proteins, salts, and other impurities are washed away with specific buffers. Finally, the DNA is eluted from the column using a low-salt buffer or water. This method is highly efficient and produces high-quality DNA suitable for most molecular biology applications. This method is fast, easy to use, highly effective at removing contaminants, and compatible with automation for high-throughput processing. Most modern DNA extraction kits are column-based (Figure 3).

Figure 3. Column-based purification of DNA.

After purification from broken proteins (enzymes, histone proteins etc.), lipids, salts, polysaccharides and phenols, DNA is resuspended in TE buffer or pure water for use.

What are the methods to measure the quantity and the quality of the extracted DNA?

Quantifying extracted DNA is essential to determine the concentration and purity of DNA samples for downstream applications like PCR, sequencing, and cloning. Some of the most commonly used methods for DNA quantification are:

• UV Spectrophotometry (NanoDrop, nanophotometer)

UV spectrophotometry is one of the most widely used methods for DNA quantification. It measures DNA concentration by assessing absorbance at 260 nm in wavelength. The purity of DNA can also be assessed by measuring absorbance at 280 and 230 to estimate the concentration of proteins and salts/organic compounds, respectively, co-extracted with the DNA.  This method is fast and simple, and it requires only a small amount of sample (as little as 1 µL). It also provides information on sample purity. However, it cannot differentiate between DNA, RNA, and free nucleotides or contaminants that absorb at similar wavelengths.

• Fluorometry (e.g., Qubit, PicoGreen, SYBR Green)

Fluorometric methods quantify DNA using fluorescent dyes that specifically bind to DNA, such as PicoGreen or Qubit reagents. The fluorescence intensity, measured by a fluorometer, is proportional to the DNA concentration in the sample. These methods are highly specific and sensitive and are less affected by contaminants compared to spectrophotometry. They require specific reagents and equipment and does not provide information on the purity or size of the DNA.

• Digital PCR (dPCR) and Real-Time Quantitative PCR (qPCR)

Digital and quantitative PCR quantify DNA by amplifying specific DNA sequences and measuring the amount of product in real time using fluorescent probes. The fluorescence intensity correlates with the DNA quantity, allowing for highly precise quantification.

These methods are highly sensitive, accurate, and can quantify very low amounts of DNA without the need for standard curves, but they are expensive, requires specialized equipment and reagents, and quantifies only specific target DNA sequences rather than total DNA.

• Capillary Electrophoresis (e.g., Bioanalyzer, TapeStation)

Capillary electrophoresis separates DNA fragments by size and measures their concentration using fluorescent dyes. The system provides quantitative data on DNA concentration, fragment size, and overall quality. These methods provide detailed information on DNA quality, size distribution, and concentration and are useful for assessing degradation and fragmentation.

• Agarose Gel Electrophoresis stained with ethidium bromide or SYBR greenorbi

The DNA is run on an agarose gel and stained with intercalating dyes like ethidium bromide or SYBR green, which fluoresce under UV or blue light. The intensity of the fluorescence of the DNA bands can be compared to a DNA ladder of known concentrations to estimate the DNA quantity. This method allows the visualization of DNA quality, size distribution, and integrity in addition to quantification. It is not very accurate for precise quantification; requires gel electrophoresis setup, staining, and imaging equipment. It is also less sensitive compared to spectrophotometry and fluorometry. The preparation of an agarose gel for DNA electrophoresis requires the use of buffers such as TAE (Tris-acetic acid-EDTA) or TBE (Tris-boric acid-EDTA).

• TBE (Tris-borate-EDTA) is a better conductive medium than TAE (Tris-acetate EDTA) and so it is less prone to overheating during long runs (> 2 h).
• Borate is an enzyme inhibitor so TBE is not a good buffer to use if you will be isolating the DNA for downstream enzymatic steps. For example, borate carry-over could affect your ligations, so use TAE instead.
• Acetate gives improved separation of large DNA fragments
• On the other hand, borate resolves <2kb fragments better, so use TBE for smaller fragments.

                         Figure 6. A) Genomic DNA on a 1% agarose gel stained with SYBR green, B) Wavelengths used to estimate DNA concentration and purity.

Molecular weight markers

Also known as DNA ladders, MWM are a mixture of DNA molecules of different lengths used in agarose or acrylamide gel electrophoresis to be used as a reference to estimate the size of unknown DNA molecules separated on the basis of their mobility in an electrical field through the gel. DNA ladders are  routinely used in every molecular biology laboratory and they are formulated for different applications. For instance, 100 bp ladders are used along the products of PCR reactions, while 1 kb ladders are preferred for genomic DNA extractions.

Figure 7. DNA weight markers or DNA “ladders”

Materials

Micropipettors set

Sterile micropipettor tips

1.7 mL Eppendorf tubes

Tubes rack

Vortex

Microcentrifuge

Cell disruptor (or tube shaking adapter for vortex)

Quick-DNA fungal/bacterial miniprep kit from Zymo Research https://www.zymoresearch.com/collections/quick-dna-fungal-bacterial-kits/products/quick-dna-fungal-bacterial-miniprep-kit

Safety concerns

This DNA extraction uses chemicals, biological materials, and equipment  that pose some potential risks:

1. Handle the ethanol and the kit reagents with gloves, at all times.
2. Use biosafety protocols to handle the bacteria samples  to prevent accidental exposure, and dispose of all the waste in a biohazard container.
3. Make sure you balance the  microcentrifuges correctly before you use them.
4. Avoid eating or drinking, and manage spills and emergencies following the directions of the instructor.

Protocol

DNA extraction from Escherichia coli cultures

1. Transfer 1.7 mL of an E. coli overnight culture into an Eppendorf tube and centrifuge at 10000 rpm for 5 min. Discard the supernatant.
2. Resuspend the cell pellet in 200 uL of 1X PBS and transfer to a ZR BashingBead Lysis tube.
3. Add 750 uL of BashingBead buffer and cap tightly.
4. Secure the tubes in a bead beater and process at maximum speed for 20 min.
5. Centrifuge the lysis tube at 10000 rpm for 1 min.
6. Transfer 400 uL of the supernatant to a Zymo-Spin III-F filter in a collection tube and centrifuge at 8000 rpm for 1 min. Discard the filter.
7. Add 1200 uL of genomic lysis buffer to the filtrate in the collection tube and mix well with a pipette.
8. Transfer 800 uL of the mixture from the previous step into a Zymo-Spin IICR column in a collection tube and centrifuge at 10000 rpm for 1 min.
9. Discard the flow through from the collection tube ad repeat the previous step. Discard the flow through.
10. Add 200 uL of DNA pre-wash buffer to the IICR column in a new collection tube and centrifuge at 10000 rpm for 1 min. Discard the flow through.
11. Add 500 uL of g-DNA wash buffer to the IICR column and centrifuge at 10000 rpm for 1 min.
12. Transfer the IICR column to a clean 1.5 mL microcentrifuge tube and add 50 uL of DNA elution buffer directly into the column matrix an incubate at room temperature 1 min. Centrifuge at 10000 rpm for 30 sec to elute the DNA.
13. Label the tube and keep frozen until use.

Preparing, pouring and running an agarose gel

1. To prepare a 1% agarose gel, weight either 0.7 or 1 g of agarose (for a small and large gel, respectively) and transfer it into a 200 mL beaker with either 70 or 100 mL of 1X TBE buffer. Heat the mixture in the microwave in 30 s increments, hand stirring the content gently with a circular motion until the agarose dissolves completely. Once The agarose is completely dissolved, add either 7 or 10 uL of SYBR safe, dissolve it and pour the mix into the gel bed and insert the comb on the negative side of the chamber (Figure 3A). Let it solidify (~20 min) or until it turns opaque.

2. Once the gel solidifies, place it in the electrophoresis chamber and pour 1X TBE buffer on both sides of the tank until the gel is covered with a ~2 mm thick buffer layer. Pull out the comb gently without breaking the walls of the wells.

3. Take the DNA samples out of the freezer and thaw them at room temperature. Mix 10 uL of the DNA with 5 uL of loading buffer on a 2 X 2 cm piece of parafilm paper, pipetting the liquids up and down twice. Skipping the first and last lanes of the gel, pipet the mix (~15 uL) into separate wells of the gel (Figure 3B). Save the first and last lanes of each gel for the DNA molecular weight marker or DNA ladder)

4. Connect the electrodes of the chamber to the power supply (make sure the DNA samples are on the negative side!) and run at 100-120 mV for 40-60 m or until the blue dye in the loading buffer reaches ¾ of the length of the gel (Figure 3C). Once the run is complete, place the gel in the transilluminator and follow the instructor directions to take a photograph for your lab report.

Figure 8. A) pouring, B) loading, and C) running a DNA agarose gel.

Lecture slides

Download the slides to follow the pre-lab lecture. DNAextraction_2025

Lab report

Download the DNA extraction lab report file and submit on Canvas, following the directions of your instructor.