How Alkaline Lysis Works

lkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since. The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes.

1. Cell Growth and Harvesting

The procedure starts with the growth of the bacterial cell culture harboring your plasmid. When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium.

2. Re-suspension

The pellet is then re-suspended in a solution (normally called solution I, or similar in the kits) containing Tris, EDTA, glucose and RNase A. Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates divalent cations in the solution preventing DNases from damaging the plasmid and also helps by destabilizing the cell wall. Glucose maintains the osmotic pressure so the cells don’t burst and RNase A is included to degrade cellular RNA when the cells are lysed.

3. Lysis

The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). This process is called denaturation and is central part of the procedure, which is why it’s called alkaline lysis. SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process.

It is important during this step to make sure that the re-suspension and lysis buffers are well mixed, although not too vigorously. Also remember that SDS and NaOH are pretty nasty so it’s advisable to wear gloves and eye protection when performing alkaline lysis.

4. Neutralization

Addition of potassium acetate (solution 3) returns decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal those huge gDNA stretches. This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.

5. Cleaning and concentration

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions.

Fruit Flies and Test Tubes Open New Window on Alzheimer's Disease

ScienceDaily (Mar. 16, 2010) — A team of scientists from Cambridge and Sweden have discovered a molecule that can prevent a toxic protein involved Alzheimer's disease from building up in the brain. They found that in test tube studies the molecule not only prevents the protein from forming clumps but can also reverse this process. Then, using fruit flies with Alzheimer's disease, they showed that the same molecule effectively "cures" the insects of the disease.

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Alzheimer's disease is the most common neurodegenerative disorder and is linked to the misfolding and aggregation of a small protein known as the amyloid β (Aβ) peptide. Previous studies in animal models have shown that aggregation of Aβ damages neurones (brain cells) causing memory impairment and cognitive deficits similar to those seen in patients with Alzheimer's disease. The mechanisms underlying this damage are, however, still not understood.

The new molecule -- designed by scientists in Sweden -- is a small protein known as an Affibody (an engineered binding protein). In this new study, researchers at the University of Cambridge and the Swedish University of Agricultural Sciences found that in test-tube experiments this protein binds to the Aβ peptide, preventing it from forming clumps and breaking up any clumps already present.

In a second experiment, they studied the effect of this Affibody in a Drosophila (fruit fly) model of Alzheimer's disease previously developed at Cambridge.

Working with fruit flies that develop the fly equivalent of Alzheimer's because they have been genetically engineered to produce the Aβ protein, they crossed these flies with a second line of flies genetically engineered to produce the Affibody.

They found that offspring -- despite producing the Aβ protein -- did not develop the symptoms of Alzheimer's disease.

According to lead author Dr Leila Luheshi of the Department of Genetics at University of Cambridge: "When we examined these flies we found that the Affibody not only prevented and reversed the formation of Aβ clumps, it also promoted clearance of the toxic Aβ clumps from the flies' brains."

"Finding a way of preventing these clumps from forming in the brain, and being able to get rid of them, is a promising strategy for preventing Alzheimer's disease. Affibody proteins give us a window into the Alzheimer's brain: by helping us understand how these clumps damage brain cells, they should help us unravel the Alzheimer's disease process."

According to Professor Torleif Härd of the Swedish University of Agricultural Sciences and one of the senior authors of the study: "Our work shows that protein engineering could open up new possibilities in Alzheimer's therapy development."

The study was supported by grants from the Swedish Research Council, the MIVAC Swedish Foundation for Strategic Research Centre, the German Academic Exchange Service, and in the UK by the MRC, the Engineering and Physical Sciences Research Council and the Wellcome Trust.