Southern Blot Hybridization Outline of Lecture n Basic steps n Purpose n Blotting methods u Mechanics n Membrane choices u Blotting solutions n Blocking agents

Basic Steps n Subject DNA fragments to agarose gel electrophoresis n Prepare the DNA in the gel for blotting n Blot the DNA to membrane such that position of fragments in gel is maintained on the membrane n Affix blotted DNA to membrane n Probe for DNA of interest on blot u Prehybridization u Hybridization with labeled probe u Rinse u Visualize

Blotting

Prehyb/Hybridization/Rinses

Blot hybridization of PCR products from several different follicular B cell lymphoma samples with MBI probe

Advantages of Southern blotting n Increases specificity of detection by incorporating two distinguishing features u Size F gel electrophoresis u Sequence F Complementary oligo- or polynucleotide probe n Increases sensitivity of fragment detection by using probe label that amplifies signal

Some Specific Purposes n To confirm that a PCR product includes a specific sequence u PCR product seen (or not seen) in gel contains expected sequence for a 14;18 translocation n To determine which fragment or fragments among the many resulting from a restriction enzyme digest contain a sequence of interest u To distinguish between a monoclonal and polyclonal population of B lymphocytes n To detect restriction fragment length polymorphisms (RFLPs) in genomic DNA

How much DNA should be run in a gel lane to allow visualization with probe? n Depends upon u the relative abundance of the target sequence to which hybridization must take place u the sensitivity of the visualization system n Current minimum = ~60fg of a bp band length u = to 60 fg of PCR product containing the sequence of interest (if using a polynucleotide probe) F can you see that with EtBr staining? u = to 120 ng of total human DNA to pick out a band of a single copy gene from a restriction digest u Radioactively labeled probe provides the greatest sensitivity, but for many applications, the sensitivity of non-radioactvely labeled probe is sufficient

Prepare the DNA in the gel for blotting n Make DNA fragments >20 kb shorter so they blot out of the gel easily (but still in place). u nick by depurination F brief exposure to.25 – 0.5 N HCl F makes sugar-phosphate backbone open to cleavage by OH -. n Denature the DNA u soak gel in alkaline solution u makes DNA single-stranded so can hybridize with probe after blotting u neutralize following denaturation if doing a neutral transfer (more later)

Blot DNA to membrane Mechanics of transfer n Capillary (no special equipment required!) u upward u downward n Electrophoretic u especially good for small fragments resolved by PAGE n Vacuum u more efficient and quantitative than capillary u must apply vacuum evenly and not too strongly

Blotting (upward)

Blotting (downward)

Transfer solutions - 3 main choices n Neutral, high ionic strength n Neutral, low ionic strength n Alkaline, low ionic strength

Transfer solution hints n Follow the membrane manufacturer’s recommendations u Alkaline blotting to charged nylon can  increased background with chemiluminescent visualization. u Some nylon membranes deteriorate with lengthy exposure to alkaline conditions u Alkaline blotting doesn’t work with nitrocellulose F DNA won’t stick above pH 9 F Alkaline conditions degrade nitrocellulose n High ionic strength buffer works for all three membrane types, but alkaline to charged nylon is most efficient if background won’t be a problem!

How long does capillary transfer take? It depends on the n Size of DNA - the longer, the longer n % of agarose in gel - the higher, the longer n Thickness of the gel - the thicker, the longer n Direction of transfer - upward takes longer u accumulating pressure compresses gel and retards diffusion n Transfer buffer - u upward alkaline transfer takes ~2 hours u upward neutral transfer takes hours

Membrane types n Charged nylon u Durable u Nylon modified with amine groups n Uncharged nylon u Durable n Nitrocellulose u Fragile u Used primarily for protein transfers

Properties of membranes

DNA fixation methods n Baking at 80 o C u DNA non-covalently, hydrophobically bonded to any membrane n Alkaline blotting u DNA covalently bonded to charged nylon membrane n UV cross-linking u DNA covalently bonded to any nylon membrane

Blocking agents n SDS n Non-fat dry milk n BSA (bovine serum albumin) n PVP n Ficoll n Proprietary commercial preparations Every manufacturer of membranes makes specific blocking recommendations. It’s best to follow those first and modify as necessary.