Southern blotting is a common method used for the study of gene organization. Current methods of DNA transfer for Southern blotting, however, can be inefficient for high concentration agarose gels. Here, we report a method for high-performance Southern blotting of short DNA fragments such as nucleosomal DNAs by using a discontinuous agarose zone gel. The results show that sharp and well-resolved fractionation of short DNA fragments comparable to that from a high-concentration agarose gel could be obtained using a low-concentration agarose gel with a small zone of high-concentration agarose, and that the resulting DNA transfer is highly efficient and rapid.

Southern blotting is routinely used to detect changes in the structure of a specific region in the genome (1). Genomic DNA is first digested with nucleases such as restriction enzyme or micrococcal nuclease. The resulting DNA fragments are separated by agarose gel electrophoresis, then denatured and transferred to a membrane for detection by hybridization with a probe. Current methods of DNA transfer mainly include upward and downward capillary transfer (2), vacuum DNA transfer (3), and alkali DNA transfer (4).

The efficiency of DNA transfer by these methods commonly depends on the length of the DNA fragments and the agarose concentration of the gel. A proper concentration of agarose in the gel (~0.7%) is usually required (5). As micrococcal nuclease has the ability to digest double-strand DNA within nucleosomal linker regions, it is used routinely to study how genes are organized in chromatin in vivo (6). In brief, cell nuclei are isolated and digested by micrococcal nuclease, the resulting DNA ladder with size multiples of ~147 bp (DNA length of nucleosome core) is fractionated on a high concentration agarose gel (1.5–2.0%) and subjected to Southern blot analysis. However, the use of high-concentration agarose gels might hinder later procedures such as DNA transfer. Polyacrylamide gels are suitable for fractionating short DNAs, but inefficient for capillary or vacuum transfer (7). Previous work has tried to resolve this problem (8) using formamide-containing agarose gels and transfer to diazobenzyl-oxymethyl (DBM) paper. However, DBM paper is not suitable for reprobing, and the use of formamide is harmful and might cause DNA denaturation during electrophoresis. To our knowledge, there has been no detailed study to improve the performance of Southern blotting of short DNA fragments fractionated in high-concentration agarose gels to maintain the sharpness and high resolution of the pattern DNA without requiring any special equipment.

In this study, we have developed a method based on discontinuous agarose zone gel (DAZG), and compared it with a standard high-concentration agarose gel for use in Southern blotting of short DNA fragments such as a nucleosomal DNA ladder. Our results show that high-performance Southern blotting on short DNA fragments can be achieved both rapidly and effectively through the new method using DAZG but not the traditional method.

The concept of DAZG is to make a low-concentration agarose gel that has the size-fractionating capacity of a higher-concentration gel. In this way, the nucleosomal DNA ladder from a micrococcal nuclease digestion can be effectively resolved and then rapidly and easily transferred. To achieve this goal, we first prepare a pair of gel spacers from used X-ray film with a thickness of ~0.2 mm. After assembling the gel-making cassette with a comb, 0.7% TAE agarose gel was poured into the gel container. Immediately, one spacer was inserted 2 cm from the wells and the second spacer inserted 1 cm below the first one before the gel was solidified. After the gel was solidified, the two spacers were taken out without disturbing the remaining gel. A razor was then used to remove the 1-cm portion of gel between the two spacers, and excess TAE buffer was wiped out. Finally, a small portion of 1.5–2% agarose gel solution was prepared and poured into the space between the two spacers (Figure 1A). After the DAZG was solidified, 1× TAE buffer was used to run the gel normally. To test the effectiveness of DAZG for resolving short DNA fragments, we used a 100-bp DNA ladder marker with six bands (600 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp). After running the DAZG, we obtained very sharp and well-separated bands for those DNA fragments (Figure 1B). Furthermore, to test whether the distance of the high-concentration agarose zone from the wells would affect the DNA fractionation, we ran two additional gels with the zone located 1cm and 3cm away from the wells. The results indicated that the distance of the high-concentration agarose zone from the wells did not affect the resolution of the short DNA fragments (Figure 1C). However, keeping the high-concentration agarose zone in the upper half of the gel and ~2 cm away from the wells and and ~5 cm from the bottom is recommended for later steps such as gel cutting.

Figure 1. DAZG preparation and comparison of the resolution of short DNA marker fragments electrophoresed on different concentration agarose gels. (Click to enlarge)

We compared the ability to resolve the short DNA fragments of different concentrations of a standard agarose gel with DAZG (Figure 1D). Using a 0.7% agarose gel, the DNA ladder marker was barely resolved, with the 100–600-bp DNA fragments stacked closely together. Using a 1.5% agarose gel, we obtained clear resolution comparable to that of DAZG.

Using nucleosomal DNA samples, we checked the ability of DAZG to enhance Southern blotting of short DNA fragments. We purified nuclei from fresh liver tissue of C57BL/6 mice and used micrococcal nuclease to digest the nuclei (9). The resulting nucleosomal DNA ladder was separated by 0.7%, 1.5% and DAZG agarose gels (Figure 2C) and the gels were subjected to DNA transfer and hybridization. Upward capillary transfer was used to transfer DNA with 10× SSC for 8 or 24 h. The probe for hybridization was designed on the basis of the restriction enzyme accessibility assay on the β-actin gene ACTB (Figure 2, A and B) using forward primer 5′-GAGACCTTCAACACCCCAGC-3′ and reverse primer 5′-AAAAGAGCCT-CAGGGCATCG-3′. Other procedures were following the standard protocol (7) for the use of Hybond XL membrane (GE Healthcare, Piscataway, NJ, USA). The radio-isotope signal detected by phosphor screen was recorded by Storm 480 (GE Healthcare). The result showed that the 0.7% gel could not adequately resolve the nucleosome DNA ladder, causing the hybridization bands to be fuzzy. While the 1.5% gel could resolve the nucleosome DNA ladder well, it resulted in a much lower hybridization signal compared with the DAZG after DNA transfer for 8h for both types of gel. This is due to the very slow capillary transfer velocity and very low amount of transfer buffer passing through the 1.5% agarose gel after 8h of transfer. Usually ≥24 h transfer time is required to overcome this problem (7); however, even then the hybridization signal from the 1.5% gel was less than that of DAZG (Figure 2D).

Figure 2. The performances of 0.7% gel, 1.5% gel, or DAZG for Southern blotting of nucleosomal DNA fragments. (Click to enlarge)

In this study, we report using DAZG is a simple, straightforward, time-saving, and high performance method to enhance sensitivity of Southern blotting on short DNA fragments such as nucleosomal DNA ladders from micrococcal nuclease digestion.