DNA and RNA Chromatography

One of the great strengths of DNA and RNA Chromatography (Chapter 12) is that UV detection can be used for the direct detection of nucleic acids. But many researchers also use fluorescence detection because of its extreme sensitivity. Fluorescence detection lowers the amount of DNA or RNA that can be detected by a factor of 10 to 100 (or even 1000 in some reported cases). 

The reader may well ask what nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have to do with ion chromatography. Nucleic acids are very large biological molecules that are more like linear polymers and bear little similarity to common anions such as chloride and sulfate. Nevertheless, DNA and RNA are anionic by virtue of the phosphate groups attached to each nucleotide sugar unit. Nucleic acids can be separated by chromatography, often by ion-pair methods similar to those described in Chapter 9. For these reasons, it seems appropriate to include a chapter on DNA and RNA chromatography in this book. 

Several column types have been used to separate nucleic acids. A discussion of the historical development of DNA and RNA chromatography is followed by a brief description of the column development that has led to the modern use of ion-pairing chromatography. 

DNA and RNA chromatography require that the instrument, column and eluent be completely free of metal contamination. The effect, source, and control of metal contamination wiU be discussed. A high-quality oven must be used. The instrument normally includes the option of collecting the nucleic acid in a fragment collector for further research and processing. 

Finally, applications of DNA and RNA chromatography wiU be shown. The purpose of using this type of methodology is to provide the means to answer biological questions. The power of the methodology is illustrated through its various ap-pUcations. 

The nondenaturing mode of DNA and RNA chromatography is used to separate double-stranded nucleic acids. This is most often used for DNA, but some short sequences of RNA can also be double-stranded. 

One of the powerful features of DNA and RNA chromatography is that material can easily be purified by collecting directly from the detector effluent Purification can be used in biological samples, where there may be several types of nucleic acids present and a particular type is desired for study. An example of this is shown later in this chapter where several types of RNA are detected in a cell extract and can be purified. 

---