Overview of DNA Extraction Methods

Abstract

DNA extraction is a fundamental method in molecular biology, despite being developed unintentionally. In 1869, the chemist Friedrich Miescher attempted to separate the cytoplasm from the nucleus in human leukocytes. He would filter suspended cells and treat them with diluted hydrochloric acid. This resulted in the discovery of a strange substance he dubbed “nuclein.” While Miescher wrongly speculated about the role of “nuclein,” his work paved the way for future developments in DNA extraction leading to techniques that are more reproducible, easier and faster to perform, cost-effective, and produce higher yields with minimal impurities.

Introduction

DNA, which is present in almost all organisms, is widely accepted as the blueprint of life. Stored within the lengthy sequences of its polynucleotides are the instructions for making every protein and molecule essential for growth, development, and health. When isolated, DNA can be used in various downstream applications, mainly polymerase chain reaction (PCR) and real-time PCR (qPCR), DNA sequencing, southern blotting, preparation of genomic libraries, and various genetic polymorphism applications. In modern medicine, many of these applications serve as the basis for prescreening and diagnosing genetic disorders and developing novel gene therapies and gene-editing technologies. Although several variations of DNA extraction have been developed since Miescher's initial discovery (figure 1), the principle behind each method consists of the same basic steps: (1) disruption of the cell and nuclear membranes; (2) separation of DNA from the cell lysate (e.g., lipids, proteins, and other nucleic acid species); and (3) concentration and purification of DNA. Determining which DNA extraction method to use is primarily influenced by the intended downstream applications for which the isolated DNA will be used. Besides the quality and quantity of the DNA extracted, other factors to consider are time, cost, yield, laboratory equipment, and the amount of starting material needed for the experiment.

Figure 1. The development of different DNA extraction techniques over the years.

DNA Extraction and Purification Methods

Phenol-Chloroform Extraction

A common way to isolate DNA is via the phenol-chloroform extraction method. This method is suitable for extracting DNA from various samples, including blood, suspension culture, and tissue. It produces relatively high yields and higher purity DNA than conventional extraction methods. Because this technique uses toxic chemicals, such as phenol, to denature proteins and chloroform to solubilize lipids, it should be performed in a fume hood, and the necessary precautions should be taken while handling.

Like all DNA extraction methods, the phenol-chloroform process begins with destroying the cell membrane and non-nucleic acid cellular components. Cells are treated with a lysis buffer typically containing denaturing detergents, such as sodium dodecyl sulfate (SDS), and depending upon the type of DNA (e.g., genomic, mitochondrial, or plasmid), can include other additives. For instance, lysis buffers for extracting plasmid DNA from bacterial cells will contain sodium hydroxide for alkaline lysis and potassium acetate for the renaturation of the plasmid DNA.

A mixture of phenol:chloroform:isoamyl alcohol (PCIA) is then added to the lysate to denature proteins and facilitate the precipitation of DNA. Since phenol is hydrophobic and less dense than water, centrifugation is used to partition the lysate into three distinct layers or phases. The bottom layer, or "organic phase," contains hydrophobic molecules like phenol, lipids, and chloroform. The middle layer, or "interphase," consists of denatured proteins, and the top layer, or "aqueous phase," comprises DNA and other polar molecules. The aqueous phase is then removed and transferred to a clean tube.

Transferring the aqueous layer requires careful pipetting. Disruption of the interphase risks exposing the aqueous phase to the organic phase and may cause contamination. When the aqueous phase is successfully transferred, DNA is precipitated using a solution of ammonium acetate and ethanol. The resulting DNA pellet is separated via centrifugation. Often, multiple ethanol washes are needed to remove contaminants and further concentrate the DNA. Each added wash involves removing the supernatant, resuspending the pellet with ethanol, and centrifuging the tube. Following the final wash, the pellet is air-dried and resuspended in a polar solution, such as an elution buffer.

Figure 2. Illustration of organic DNA extraction using the phenol:chloroform:isoamyl alcohol (PCIA) method. PCIA partitions DNA to the aqueous phase while lipids and proteins are partitioned into the organic and interphases, respectively.

Silica-Phase Extraction

Besides organic methods, solid-phase extraction using a solid substrate, such as silica resins or beads, is another popular way to isolate DNA. Instead of using solvents to force DNA precipitation, this technique uses a simple lyse-bind-wash-elute process. First, cells are lysed with a buffer solution containing 1% SDS, 0.05 M EDTA, 0.2 M Tris pH 8.0, and Proteinase K. A binding buffer made up of various chaotropic salts (e.g., phenol, ethanol, guanidine hydrochloride, urea) is added to the lysate and then transferred to a spin-column and centrifuged. Centripetal forces push the solution through the silica membrane within the spin column, whereby DNA binds to the silica membrane. The rest of the solution (e.g., proteins and organic phase) passes through the column and is discarded.

The spin column undergoes multiple washes with various buffers, each formulated to remove specific contaminants while maintaining DNA binding conditions. Following the last wash step, DNA is selectively eluted under low-salt conditions using TE buffer.

Both techniques will yield high-quality DNA samples, but there are optional treatments to improve sample quality. Enzymes such as proteases and nucleases can be added to the sample to remove cellular contaminants further. Some examples are Proteinase K and various RNases. The use of enzymatic degradation does add to the overall cost, but the increased sample quality often makes it worth it.

Figure 3. Illustration of solid-phase DNA extraction using silica gel spin columns. In the presence of chaotropes, DNA binds to silica resins while other cellular components are washed away.

Quantitative and Qualitative Analysis of DNA Yield and Purity

Following successful DNA extraction and before the intended downstream application, the yield and purity of the DNA must be assessed. The primary methods to measure DNA yield and purity include absorbance, fluorescence, and gel electrophoresis.

Of the three, absorbance methods are the most common and easiest. They do not require additional reagents like fluorescence-based methods, which need DNA-selective dyes. All that is required is a spectrophotometer. Absorbance readings of the DNA solution are taken at 260 nm, where DNA absorbs light maximally, and at 230, 280, and 320 nm, which correspond to absorbance peaks of common chemical contaminants or chaotropic salts, proteins, and turbidity, respectively. The number recorded at A₂₆₀ can be used to estimate the concentration of the solution and should be within the linear range of the instrument (0.1-1.0). Based on the relationship that A₂₆₀ of 1.0 is equivalent to 50 µg/mL pure dsDNA, the following formula can be used to estimate DNA concentration:

Concentration (µg/mL) = (A₂₆₀ reading - A₂₃₀ reading) x dilution factor x 50 µg/mL

To determine the total yield, take the calculated DNA concentration and multiply it by the final sample volume:

DNA yield (µg) = DNA concentration (µg/mL) x total sample volume (mL)

To evaluate DNA purity, calculate absorbance ratios at A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀. For pure DNA samples, the A₂₆₀/A₂₈₀ ratio should be between 1.8-2.0. The ratio A₂₆₀/A₂₃₀ is used to evaluate the level of organic compounds present in the purified DNA and should range between 2.0-2.2.

A downside to measuring absorbance is that other nucleic acids also peak at 260 nm. The listed concentration from the 260 nm reading cannot differentiate between nucleic acid types in the sample. The concentration could be just DNA or a mix of DNA, RNA, and other nucleic acid species. A workaround is to use DNA-sensitive fluorophores such as Helixyte™ Green. These dyes selectively bind to double-stranded DNA and, when excited, emit strong fluorescence signals proportional to the amount of dsDNA present. Although fluorescence-based methods offer greater sensitivity, specificity, and wider dynamic ranges than absorbance methods, they cannot provide information about contamination or extraction quality.

Protocol for DNA Extraction and Purification

Purpose

The phenol-chloroform extraction is a straightforward method to isolate DNA from the lysate. This technique relies on chemical means to separate DNA from the lysate. Though it is more time-consuming than spin column extraction, phenol-chloroform extraction can yield high-quality DNA with minimal reagent cost.

Materials

• Ammonium acetate, concentrated solution (5M - 7.5M)
• 25:24:1 phenol/chloroform/isoamyl alcohol solution, pH 7.8-8.2
• 24:1 chloroform/isoamyl alcohol solution
• Elution buffer (10 mM Tris-HCl, pH 8.5)
• Glycogen, 20 mg/mL (molecular biology grade)
• 100% Ethanol
• 80% Ethanol

Procedure

1. Add 200 µL of starting material (lysate) into Tube 1. Bring the total volume to 200 µL with the elution buffer if needed.
2. Add 200 µL of the phenol/chloroform/isoamyl alcohol solution into Tube 1.
3. Vortex Tube 1 for 30-60 seconds.
4. Centrifuge Tube 1 at 16,000xg for 5 minutes at room temperature.
5. Remove the top aqueous layer from Tube 1 and transfer it into a new clean tube, Tube 2.
6. Add 200 µL elution buffer into Tube 1.
7. Repeat steps 3 and 4.
8. Remove the top aqueous layer from Tube 1 and transfer it to Tube 2.
9. Add equivalent volumes of the 24:1 chloroform/isoamyl alcohol solution into Tube 2.
10. Vortex Tube 2 for 30-60 seconds.
11. Centrifuge Tube 2 at 16,000xg for 5 minutes at room temperature.
12. Remove the top aqueous layer from Tube 2 and transfer it into a new clean tube, Tube 3.
13. Add ammonium acetate to Tube 3 until the final concentration is 0.75 M.
14. Add 1 µL of the glycogen mixture into Tube 3. Pipette up and down repeatedly to mix.
15. Add 100% ethanol at 2.5 times the volume into Tube 3.
16. Centrifuge Tube 3 at max force for 20 minutes at 4°C.
17. Remove the supernatant without disturbing the pellet.
18. Wash by adding 300 µL of 80% ethanol into Tube 3. Pipette up and down to resuspend the pellet.
19. Centrifuge tube at max force for 15 minutes at 4°C
20. Remove the supernatant without disturbing the pellet.
21. Repeat steps 15 to 17 for a second 80% ethanol wash.
22. Spin Tube 3 on a tabletop centrifuge and remove residual ethanol with a P20 pipette.
23. Let air dry for several minutes
24. Dissolve the pellet in elution buffer or DNase-free molecular-grade water
    1. Keep on ice if measuring concentration in the sample. Otherwise, store at -20°C. Avoid multiple freeze-thaw cycles to preserve sample integrity

References

1. Barnett, R., & Larson, G. (2012). A phenol-chloroform protocol for extracting DNA from ancient samples. Methods Mol Biol, 840, 13-19. https://doi.org/10.1007/978-1-61779-516-9_2
2. Gahlon, H. L. (2020). A Brief History and Practical Applications in DNA Extraction. Chimia (Aarau), 74(11), 907-908. https://doi.org/10.2533/chimia.2020.907
3. Gupta, N. (2019). DNA Extraction and Polymerase Chain Reaction. J Cytol, 36(2), 116-117. https://doi.org/10.4103/JOC.JOC_110_18
4. Sun, W. (2010). Nucleic Extraction and Amplification. In Molecular Diagnostics (pp. 35-47). https://doi.org/10.1016/b978-0-12-369428-7.00004-5