DNA and RNA are the key information-rich molecules in cells, comprising nearly a quarter of the dry weight of E. coli, for example. Methods for extracting and purifying nucleic acids play a central role in contemporary biosciences because nucleic acids are intimate participants in the storage and retrieval of hereditary information.
Extraction procedures lyse cells and prepare nucleic acids for subsequent analysis, such as end-point PCR, which does not require highly pure starting material. Purification procedures, in contrast, remove proteins, lipids, carbohydrates, and cell debris, leaving concentrated nucleic acids suitable for cloning, sequencing, Northern or Southern blotting, RT-PCR, and microarray analysis. Because purification precedes further often complex downstream analysis, it is essential to invest some thought and effort into one’s purification methods and reagents.
The three most common purification technologies are magnetic beads or particles, spin columns with disks that adsorb nucleic acids, and selective precipitation. A few advantages and disadvantages of these methods are summarized in the Table.
Spin columns are the most commonly used technology, although for robotic high-throughput applications, magnetic beads are easily adaptable to and are popular for 96-well plate formats. On the other hand, the total yield from spin columns is lower than for selective precipitation, and high molecular weight molecules are not efficiently released from spin column adsorbent disks. Selective precipitation requires more centrifugation steps but delivers higher yields.
The purification technology chosen depends ultimately on the needs of the user. If the goal is to obtain some nucleic acid from dozens or hundreds of samples, for example, then a rapid or high-throughput procedure is warranted. If, on the other hand, high molecular weight nucleic acids are desired, then it would be wise to use a slower, but more robust method for retaining larger size classes of molecules.