Complete disruption and lysis of cell walls and plasma membranes of cells and organelles is an absolute requirement for all genomic DNA isolation procedures. Incomplete disruption results in significantly reduced yields.
Disruption generally involves use of a lysis buffer that contains a detergent (for breaking down cellular membranes) and a protease (for digestion of protein cellular components). The choice of protease depends on the lysis buffer used. Some sample types require additional treatment for efficient lysis; this is described in more detail in Special considerations for isolating genomic DNA from different sample sources.
Disruption using rotor–stator homogenizers
Rotor–stator homogenizers thoroughly disrupt animal and plant tissues in 5–90 seconds depending on the toughness of the sample. The rotor turns at very high speed causing the sample to be disrupted by a combination of turbulence and mechanical shearing. Foaming of the sample should be kept to a minimum by using properly sized vessels, by keeping the tip of the homogenizer submerged, and by holding the immersed tip to one side of the tube. Rotor–stator homogenizers are available in different sizes and operate with probes of different sizes. Probes with diameters of 5 mm and 7 mm are suitable for volumes up to 300 µl and can be used for homogenization in microfuge tubes. Probes with a diameter of 10 mm or above require larger tubes.
Disruption using bead mills
In disruption using a bead mill, the sample is agitated at high speed in the presence of beads. Disruption occurs by the shearing and crushing action of the beads as they collide with the cells. Disruption efficiency is influenced by:
Size and composition of beads
Ratio of buffer to beads
Amount of starting material
Speed and configuration of agitator
The optimal beads to use are 0.1 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, 3–7 mm stainless steel beads for animal tissues, and 3–7 mm stainless steel or tungsten carbide beads for plant and fungal tissues. It is essential that glass beads are pretreated by washing in concentrated nitric acid. Alternatively, use commercially available acid-washed glass beads. All other disruption parameters must be determined empirically for each application.
Disruption using a mortar and pestle
For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen–cooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the isolation procedure.
Special considerations for isolating genomic DNA from different sample sources
Some sample sources contain substances that can cause problems in DNA isolation and analysis. Special considerations are required when working with these sample sources. In this section, considerations for working with a number of different sources are discussed.
Human blood samples are routinely collected for clinical analysis. Blood contains a number of enzyme inhibitors that can interfere with downstream DNA analysis. In addition, common anticoagulants such as heparin and EDTA can interfere with downstream assays. DNA isolation from blood requires a method to provide high-quality DNA without contaminants or enzyme inhibitors.
In animals, erythrocytes (red blood cells) from birds, fish, and frogs contain nuclei and hence genomic DNA, while those from mammals do not. Since healthy mammalian blood contains approximately 1000 times more erythrocytes than nuclei-containing leukocytes (white blood cells, comprising lymphocytes, monocytes, and granulocytes) removing the erythrocytes prior to DNA isolation can give higher DNA yields. This can be accomplished by several methods. One is selective lysis of erythrocytes, which are more susceptible than leukocytes to hypotonic shock and burst rapidly in the presence of a hypotonic buffer. Alternatively, Ficoll density-gradient centrifugation can be performed to recover mononuclear cells (lymphocytes and monocytes) and remove erythrocytes. This technique also removes granulocytes. A third method is to prepare a leukocyte-enriched fraction of whole blood, called buffy coat, by centrifuging whole blood at 3300 x g for 10 minutes at room temperature. After centrifugation, three different fractions are distinguishable: the upper clear layer is plasma; the intermediate layer is buffy coat; and the bottom layer contains concentrated erythrocytes.
Blood samples, including those treated to remove erythrocytes, can be efficiently lysed using lysis buffer and protease or proteinase K. Along with the animal’s genomic DNA, viral and bacterial DNA can also be isolated from blood samples.
Other clinical samples
Most biological fluids can be treated in the same way as blood samples for isolation of DNA. Isolation of DNA from stool samples is more difficult, as stool typically contains many compounds that can degrade DNA and inhibit downstream enzymatic reactions.
Animal tissues and cell culture
Animal cell cultures and most animal tissues can be efficiently lysed using lysis buffer and protease or proteinase K. Fresh or frozen samples should be cut into small pieces to aid lysis. Mechanical disruption using a homogenizer or mortar and pestle prior to lysis can reduce lysis time. Skeletal muscle, heart, and skin tissue have an abundance of contractile proteins, connective tissue, and collagen, and care should be taken to ensure complete digestion with protease or proteinase K.
For fixed tissues, the fixative should be removed prior to lysis. Formalin can be removed by washing the tissue in phosphate-buffered saline (PBS). Paraffin should be similarly removed from paraffin-embedded tissues by extraction with xylene followed by washing with ethanol.
Yeast cell cultures
Yeast cell cultures must first be treated with lyticase or zymolase to digest the cell wall. The resulting spheroplasts are collected by centrifugation and then lysed using lysis buffer and proteinase K or protease.
Many bacterial cell cultures can be efficiently lysed using lysis buffer and protease or proteinase K. Some bacteria, particularly Gram-positive bacteria, require pre-incubation with specific enzymes (e.g., lysozyme or lysostaphin) to lyse the rigid, multilayered cell wall.
Bacterial DNA can also be isolated from a wide variety of clinical samples. Bacterial cells should be pelleted from biological fluids, and the DNA isolated as for bacterial cell cultures. Swab samples should be pretreated with fungicide before centrifugation of bacterial cells.
In clinical applications, viral DNA is often (although not always) isolated from cell-free body fluids, where their titer can be very low. Virus particles may need to be concentrated before DNA isolation by ultracentrifugation, ultrafiltration, or precipitation. Addition of carrier DNA may also be necessary during DNA isolation when the expected yield of DNA is low. Integrated viral DNA is prepared using the same procedure as for isolation of genomic DNA from the relevant sample. Bacteriophage, such as M13 and lambda, are isolated from infected bacterial cultures. The bacterial cells must be removed from the culture by centrifugation prior to isolation of viral DNA.
Isolation of DNA from plant material presents special challenges, and commonly used techniques often require adaptation before they can be used with plant samples. Several plant metabolites have chemical properties similar to those of nucleic acids, and are difficult to remove from DNA preparations. Co-purified metabolites and contaminants introduced by the purification procedure, such as salts or phenol, can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and gel migration.
DNA isolation is often improved by using plants grown under conditions that do not induce high levels of plant metabolites. Because of the great variation among plants, it is difficult to make general statements about growth conditions to use. However, as a general guideline, it is recommended to use healthy, young tissues when possible. DNA yields from young tissues are often higher than from old tissue because young tissue generally contains more cells than the same amount of older tissue. Young tissue of the same weight also contains fewer metabolites. In addition, many protocols for “home-made” DNA isolation methods recommend growing plants in darkness for 1–2 days before harvesting to prevent high-level accumulation of plant metabolites.