Overview

Southern blot analysis, initially developed in 1975 by Sir Edwin Southern, is a procedure to reveal the identity, size, and abundance (copy number) of a DNA sequence.¹ Steps of Southern blot analysis involve restriction enzyme digests of DNA samples, agarose gel electrophoresis, DNA transfer from the gel onto a membrane, probe synthesis, and nucleic acid hybridization and detection. Although Southern blot is no longer used extensively, its steps and underlying principles apply to a wide variety of research in modern biology. For example, restriction digest and agarose gel electrophoresis, one of the most fundamental techniques in molecular biology and genetics, is indispensable for restriction mapping and DNA subcloning.² Probe labeling, nucleic acid hybridization, and detection are used in various research and applications, including screening specific DNA sequences from libraries,³ probe labeling for fluorescence in situ hybridization (FISH) to detect and localize specific DNA,⁴ and identifying bacterial and eukaryotic pathogens.⁵

The prerequisite step of Southern blotting is to obtain a good quantity of high-quality genomic DNA as starting material. Depending on the genome size of the organism, the amount of DNA needs to be sufficient to detect a single copy gene (~10 µg for plants), which may be limited by the amount of DNA that can be loaded onto and separated in an agarose gel, and further affected by the sensitivity of the detection technique. While detecting single/low-copy number target sequences in a large genome can be challenging, high sensitivity with little or no background is essential to successful Southern blots.⁶

To confirm the success of degenerate PCR cloning completed in Module 1, a “plasmid-to-plasmid” Southern blot⁷ is implemented to validate the presence and location of cloned NBS sequences in recombinant plasmids. This lab module aims to provide you with hands-on experience in Southern blot analysis. The first step is to obtain the cloned recombinant plasmid from an overnight bacterial culture. In addition to being prepared for outsourcing DNA sequencing, the isolated plasmid and a known grape NBS-containing plasmid, pSCA7 (T1-T3-W6),⁸ are digested with restriction enzymes (Week 1) and separated by agarose gel electrophoresis, the gel is then photographed (Week 2). The DNA band from the previously cloned recombinant plasmid corresponding to a known NBS sequence from the pSCA7 (T1-T3-W6) is then excised and extracted from the gel for PCR Digoxygenin (DIG) probe synthesis (Week 3). The rest of the DNA in the gel is transferred onto a membrane (Week 2), which is hybridized with the labeled DIG probe, followed by washing and detecting the NBS sequence in the cloned plasmid (Week 4). Results from agarose gel electrophoresis and the Southern blot analysis are used for restriction mapping. The sequence of the DNA insert in the cloned plasmid is analyzed to validate its identity. The timeline and outline of weekly lab exercises are summarized in Figure 2-1.