DNA ISOLATION AND PURIFICATION

DNA ISOLATION AND PURIFICATION

DNA isolation methods

Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. Removal Of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology). DNA is usually recovered by precipitation using ethanol or isopropanol. The choice of a method depends on many factors: the required quantity and molecular weight of the DNA, the purity required for downstream applications, and the time and expense. Several of the most commonly used methods are detailed below, although many different methods and variations on these methods exist. Home-made methods often work well for researchers who have developed and regularly use them. However, they usually lack standardization and therefore yields and quality are not always reproducible. Reproducibility is also affected when the method is used by different researchers, or with different sample types.

The separation of DNA from cellular components can be divided into four stages:

1. Disruption

2. Lysis

Cell walls and membranes must be broken to release the DNA and other intracellular components. This is usually accomplished with an appropriate combination of enzymes to digest the cell wall (usually lysozyme) and detergents to disrupt membranes. We use the ionic detergent Sodium Dodecyl Sulfate (SDS) at 80˚C to lyse E. coli.

3. Removal of proteins and contaminants

RNA is usually degraded by the addition of RNase. The resulting oligoribinucleotides are separated from the high molecular weight (HMW) DNA by exploiting their differential solubilities in non-polar solvents (usually alcohol/water).

Proteins are subjected to chemical denaturation and/or enzymatic degradadtion. The most common technique of protein removal involves denaturation and extraction into an organic phase consisting of phenol and chloroform.

Another widely used purification technique is to band the DNA in a CsCl density gradient using ultracentrifugation.

4. Recovery of DNA

In some methods, stages 1 and 2 are combined.

Reference:

www1.qiagen.com/literature/brochures/Gen_DNA_Pur/1019469_BROS_DNYTi_p10_13.pdf

http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf