Genomic DNA isolation by the traditional phenol/chloroform method is toxic, time-consuming, and utilizes protease digestion, organic solvent extraction, alcohol precipitation, as well as centrifugation steps (1). This multistep method is inconvenient when genotyping large numbers of samples (e.g., when characterizing transgenic mice). To simplify genotyping, we developed a new DNA isolation method that avoids the phenol/chloroform purification steps and eliminates multistep tube changes. A step-by-step procedure for our ethanol method is summarized in Table 1.

Table 1. Protocol for Genomic DNA Isolation from Mouse Tails (Click to enlarge)

To compare the DNA quality resulting from the ethanol method with the standard phenol/chloroform method (1), we extracted DNA from the same set of samples using the two methods. After the tissue samples were digested overnight (Table 1), half of the resulting digestion mixture from each tube was used for the ethanol method and the other half for the standard phenol/chloroform method. Twenty microliters of DNA resuspended in Tris EDTA (TE) buffer, prepared from each sample, were electrophoresed through a 0.8% agarose gel running in TAE buffer [0.4 M Tris-base, 11.4% (v/v) glacial CH₃COOH, 10 mM disodium EDTA, pH 7.6, with glacial CH₃COOH or Tris] at 2 V/cm (Figure 1A). DNA extracted by the ethanol method and the phenol/chloroform method had identical molecular weights. Furthermore, no DNA degradation was detected in either method, indicating that both gave high quality DNA.

Figure 1. Comparison of DNA extracted with the ethanol and phenol/chloroform methods. (Click to enlarge)

We also compared standard PCR amplifications of DNA prepared by both methods (Figure 1B). We selected two chromosome loci to amplify in mouse, type III adenylyl cyclase (AC3) (2) and Gαi2 protein (3). The primers used for these two loci are listed in Table 2. We used 5 L DNA, about 50 ng, as templates in a 25-L PCR volume. The PCR amplifications were performed in a thermal Mastercycler® gradient (Eppendorf Scientific, Westbury, NY, USA) as follows: 5 min at 94°C, 40 cycles of 45 s at 94°C, 1 min at 56°C, and 1 min 30 s at 72°C, followed by a 7-min hold at 72°C. With the two extraction protocols, we were able to identify, by PCR amplification, the transgenic animals (Figure 1B). Successful amplifications were also possible when using 1 L DNA as template or fewer cycles of PCR (data not shown). This indicates that the DNA from the new method is suitable for PCR applications.

Table 2. Primers Used in PCR Amplifications (Click to enlarge)

We also examined the quality of the DNA extracted through the ethanol method by restriction endonuclease digestion. Five micrograms DNA were digested with EcoRI and BamHI (both from New England Biolabs, Ipswich, MA, USA) overnight at 37°C. The products were electrophoresed on a 0.8% agarose gel. The DNA obtained by the ethanol method was completely digested and comparable to the DNA isolated by the phenol/chloroform method (Figure 1C). This suggests that the DNA isolated by the ethanol method is suitable for Southern blot analysis.

The A260/A280 ratio is often used as a measure of nucleic acid purity. We found no differences in the A260/A280 ratio (1.2 to 1.6) when comparing the ethanol and phenol/chloroform methods (data not shown), suggesting equal amounts of protein or other contaminants.

Our new method has many advantages compared with the traditional procedure. Because the entire procedure is performed in the same tube, the yield of DNA is superior to other methods (Figure 1 A) (the mean yield is about 26 g, n = 5). We have successfully used the ethanol extraction protocol to extract DNA from over 1000 mouse samples in our laboratory. With this method, one can routinely isolate approximately 200 samples/day from mouse tissues. The simplicity of the procedure and the quality of DNA obtained by the ethanol extraction method are comparable to that obtained with various commercial kits and considerably less expensive. In addition, this procedure has been used to extract quality DNA from bird blood (data not shown; note that nuclei are retained in avian erythrocytes), indicating that the method is applicable to tissue from other species.

Other simplified methods for extracting DNA from mouse tissues are available (4,5,6,7,8). However, the DNA obtained by the HotSHOT method (4) is extensively sheared, and its concentration is often too low. Thus, HotSHOT DNA preparations are likely to be limited to PCR applications and are not suitable for genotyping transgenic mice by Southern analysis. There is a potent inhibitor of Taq DNA polymerase in the DNA extracted by the salt-out method (9). This often produces unreliable results because of the high frequency of false negatives and the inconsistent performance among different experiments (9). Other methods require tube changes (6), adding proteinase K at intervals of several hours (7), or pulverizing the tails with a plastic pestle (8). All of this is very inconvenient when genotyping large numbers of samples.

In summary, we have developed a simple, rapid, and inexpensive method for the isolation of genomic DNA from mouse tails that gives good yields and high quality DNA. The DNA obtained by this efficient and economical method is of sufficient quality for PCR amplification and restriction endonuclease digestion and therefore is expected to be applicable to Southern hybridization, library construction, and other downstream applications of PCR.