Southern Blot HybridizationOutline of Lecture

Basic steps Purpose Blotting methods

Mechanics Membrane choices

Blotting solutions Blocking agents

Basic Steps Subject DNA fragments to agarose gel electrophoresis Prepare the DNA in the gel for blotting Blot the DNA to membrane such that position of

fragments in gel is maintained on the membrane Affix blotted DNA to membrane Probe for DNA of interest on blot

Prehybridization Hybridization with labeled probe Rinse 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

Increases specificity of detection by incorporating two distinguishing features Size

gel electrophoresis Sequence

Complementary oligo- or polynucleotide probe

Increases sensitivity of fragment detection by using probe label that amplifies signal

Some Specific Purposes

To confirm that a PCR product includes a specific sequence PCR product seen (or not seen) in gel contains

expected sequence for a 14;18 translocation To determine which fragment or fragments among

the many resulting from a restriction enzyme digest contain a sequence of interest To distinguish between a monoclonal and polyclonal

population of B lymphocytes 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? Depends upon

the relative abundance of the target sequence to which hybridization must take place

the sensitivity of the visualization system

Current minimum = ~60fg of a 500-1000 bp band length = to 60 fg of PCR product containing the sequence of interest (if using a

polynucleotide probe) can you see that with EtBr staining?

= to 120 ng of total human DNA to pick out a band of a single copy gene from a restriction digest

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

Make DNA fragments >20 kb shorter so they blot out of the gel easily (but still in place). nick by depurination

brief exposure to .25 – 0.5 N HCl makes sugar-phosphate backbone open to cleavage by

OH-. Denature the DNA

soak gel in alkaline solution makes DNA single-stranded so can hybridize with

probe after blotting neutralize following denaturation if doing a neutral

transfer (more later)

Blot DNA to membrane Mechanics of transfer

Capillary (no special equipment required!) upward downward

Electrophoretic especially good for small fragments resolved by

PAGE Vacuum

more efficient and quantitative than capillary must apply vacuum evenly and not too strongly

Blotting (upward)

Blotting (downward)

Transfer solutions - 3 main choices Neutral, high ionic strength

Neutral, low ionic strength

Alkaline, low ionic strength

Transfer solution hints Follow the membrane manufacturer’s recommendations

Alkaline blotting to charged nylon can increased background with chemiluminescent visualization.

Some nylon membranes deteriorate with lengthy exposure to alkaline conditions

Alkaline blotting doesn’t work with nitrocellulose DNA won’t stick above pH 9 Alkaline conditions degrade nitrocellulose

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 Size of DNA - the longer, the longer % of agarose in gel - the higher, the longer Thickness of the gel - the thicker, the longer Direction of transfer - upward takes longer

accumulating pressure compresses gel and retards diffusion Transfer buffer -

upward alkaline transfer takes ~2 hours upward neutral transfer takes 12-24 hours

Membrane types Charged nylon

Durable Nylon modified with amine groups

Uncharged nylon Durable

Nitrocellulose Fragile Used primarily for protein transfers

Properties of membranes

DNA fixation methods

Baking at 80oC DNA non-covalently, hydrophobically bonded to any

membrane Alkaline blotting

DNA covalently bonded to charged nylon membrane UV cross-linking

DNA covalently bonded to any nylon membrane

Blocking agents

SDS Non-fat dry milk BSA (bovine serum albumin) PVP Ficoll Proprietary commercial preparations

Every manufacturer of membranes makes specific blocking recommendations. It’s best to follow those first and modify as necessary.