This kit contains materials for six groups to perform Southern transfer and hybridization analysis using the included lambda DNA samples and biotinylated probe. The intellectual objective of the experiment is to determine the region of the genome from which the probe sequence is taken. Southern hybridization analysis requires several steps. Consult this manual to plan your laboratory sessions. Some of the introduction to this kit was excerpted with permission from Recombinant DNA and Biotechnology: A Guide for Teachers by Kreuzer and Massey (2001; ASM Press, Washington, DC; Carolina Biological Supply catalog #RN-21-2218).

Introduction

During many procedures, including DNA library screening, DNA-based disease diagnosis, and DNA fingerprinting, it is important to know whether a specific DNA sequence is present in a DNA sample and where it is located with respect to restriction enzyme sites. Restriction enzyme digestion, electrophoresis, and staining allow us to cut DNA molecules into reproducible pieces and to determine the size of these pieces. However, restriction enzyme analysis alone does not provide information about DNA sequences present within the fragments. Southern hybridization analysis combines restriction enzyme analysis and hybridization analysis to provide this kind of information. Combining these techniques reveals which fragments from a restriction digest (if any) contain a specific DNA sequence. In brief, hybridization analysis involves separating (denaturing) the strands of the DNA molecules to be analyzed and then mixing those separated strands with many copies of a single-stranded DNA or RNA molecule, called a probe. The probe contains a sequence complementary to the nucleotide sequence of interest. When a probe is mixed with singlestranded (denatured) DNA under the right conditions, hydrogen bonds form between the probe and its complementary sequence in the DNA sample being analyzed. The formation of hydrogen bonds between two complementary strands to create a double-stranded complex is called hybridization, or annealing. When a DNA or RNA probe bonds to its complementary sequence in the DNA being analyzed, the probe is said to be annealing (or hybridizing) to the sample DNA. Hybridization analysis can be performed either in solution or with one component attached to some kind of solid support. For Southern hybridization analysis, the sample DNA is attached to a solid support, such as a nitrocellulose or nylon membrane.

Hybridization involves several steps. First, the probe and sample DNA are allowed to hybridize under the appropriate conditions. The correct temperature, incubation times, and buffer conditions must be used. Next, the sample DNA is washed using conditions that will remove unhybridized probe but not the hybridized probe. Finally, the sample DNA is tested for the presence of the hybridized probe. The probe is labeled with a radioactive molecular tag (or some other tag) that allows it to be detected following hybridization.

To begin Southern hybridization analysis, the sample DNA is digested with restriction enzymes and the resulting fragments are separated by agarose gel electrophoresis. The DNA must then be transferred from the agarose gel to a solid support prior to hybridization. In 1975, a scientist named Southern published a method for transferring DNA fragments from an

agarose gel to a membrane in a manner that preserved the arrangement of the fragments as they existed in the gel. Because of the scientist’s name, this transfer method is known as Southern transfer or Southern blotting. To perform Southern transfer, the agarose gel is first soaked in a basic solution to denature the DNA fragments. After an additional soaking step to neutralize the base, the gel is placed on a long piece of blotting paper with the ends of the paper suspended in a reservoir of salt solution. A nitrocellulose or nylon membrane is then laid directly on top of the gel. Blotting paper and a stack of dry absorbent paper (such as paper towels) are then placed on the membrane (see Fig. 2, page 14). The blotting paper acts like a wick. Driven by capillary action, fluid is drawn from the reservoir up though the gel and into the stack of dry paper.

As the fluid migrates up through the gel, it carries the denatured DNA fragments up with it out of the gel. When the fragments reach the membrane, they stick to the membrane and remain there. Because the wick, gel, membrane, and stack of paper lay directly on top of each other, when the DNA fragments are transferred up onto the membrane, they form the same pattern that they formed in the gel. After the transfer is complete, the DNA-containing membrane is rinsed and the denatured DNA molecules are fixed to it through heating or exposure to ultraviolet light. This membrane, with the fixed, single-stranded DNA, is now ready for hybridization with the desired probe.