Nucleic acids, separation

Many of the same models and techniques have been used to study the transitions in these two types of biopolymers, and we will present some common background information first. Then we will specialize and present the results of important thermodynamic studies in proteins and nucleic acids separately. However, common to both reports is the observation that the application of thermodynamic measurements and a thermodynamic analysis to carefully but widely chosen systems allows one to gain insights into structural details that complement molecular structure determinations obtained from instrumental techniques such as spectroscopy and X-ray crystallography. 

Reviews of the biochemical work generally start with Martin and Synge in 1941 (13) and then jump to the work of Cohn on nucleic acid separations by ion exchange chromatography in 1949 (14). It so happens that Waldo Cohn was coauthor of one of those original publications on rare earth separations in 1947. 

The separation of nucleic acids by CE has become a steadily growing area of interest, especially since the inception of the Human Genome Initiative. This interest originally stemmed from the use of polyacrylamide (PA) or agarose slab gel electrophoresis as the accepted standard for nucleic acid separation (Stellwagen, 1987). 

Though chemically similar, DNA and RNA differ in size and have different roles within the cell. Molecules of DNA are enormous. They have molecular weights of up to 150 billion and lengths of up to 12 cm when stretched out, and they are found mostly in the nucleus of cells. Molecules of RNA, by contrast, are much smaller (as low as 35,000 in molecular weight) and are found mostly outside the cell nucleus. We ll consider the two kinds of nucleic acids separately, beginning with DNA. 

The apparatus shown in Figure 27-18 is one type of a commercial design for vacuum blotting. The manufacturer says it is "Designed to handle all types of nucleic acid separations, the precision controlled vacuum pump maintains the exact vacuum level needed to blot all lengths of nucleic acids, from depurinated RNA or DNA fragments to chromosome size. No wicks, cassettes or stacks of filter paper are needed. It can do up to 10 transfers a day with close to 100% DNA recovery."... 

Clinical and biochemical. Separation of amino acids and peptides is regularly carried out to aid in the investigation of protein structures. Routine examination of urine and other body fluids for amino acids and sugars (this is most important, as it can be used for diagnosis of a number of pathological conditions, with the standard map technique). Separation of purine bases and nucleotides in the examination of nucleic acids. Separation of steroids. 

How are nucleic acids separated Two of the primary necessities for successful experiments with nucleic adds are to separate the components of a mixture and to detect the presence of nucleic acids. DNA can be cut into pieces with restriction enzymes and then separated with gel electrophoresis. 

Several other modifications of cellulose have been used to examine the retention parameters of various nucleic acid samples. For specific applications a nitrocellulose phase (61), a mercurial cellulose (62). and a sulihydryl-ccllulose (6J) were examined in separating RN A. It is evident that the mixed properties of these supports increase the selectivity for specific nucleic acid separations. 

The need for a large pore size drives us to a pore size of >0.1 pm, the desire for maximal efBdency and short analysis times drives us to a particle size of < 1 pm. Both effects cooperate to make nonporous particles of a size 1pm or slightly less an attractive option for the chromatography of macromolecules. The exact point where both effects meet is a question of both the size and the shape of the macromolecule. Indeed, careful studies (7-10) have demonstrated that small nonporous particles perform well for protein and nucleic acid separations. 

High-performance SEC columns for nucleic acid separation... 

Recently, a total serum protein N-glycosylation profiling was attempted on a CE chip by Ehrlich and coworkers [213]. The authors employed a glass chip with a double-tee injector and a 4% linear polyacrylamide sieving medium (analogous to nucleic acid separations). Profiling of serum samples from chronic hepatitis patients identified the differences in N-glycan composition in cirrhotic and noncirrhotic cases, and demonstrated the potential of microchip approach for these types of clinical studies. 

Zabzdyr, J.L. and LiUard, S.J., UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis, J. Chromatogr. A, 911, 269, 2001. 

During the past 10 years, the concepts of miniaturization have been successfully applied to chemical and biological problems. The development and application of microfluidic or lab-on-a-chip technology has been of particular interest. A few studies are developed on earth for future use in space considering the mass reduction benefit of the microscale [1,2]. But at this stage, the main difficulty is to understand fluid behavior inside the lab-on-a-chip when biological or chemical reactions occur. On earth microfluidic systems have been used in a wide variety of applications including nucleic acid separations, protein efficiency in respect to the... 

Chandler DP and Brockman FJ (2000) Renewable microcolumns for solid phase nucleic acid separations and analysis from environmental samples. Trends in Analytical Chemistry 19 314-321. 

This review gives an overview of current advances in nucleic acid separation by CE and microchip electrophoresis. The focus is on the separation mechanisms during CE, conventional separation matrices and thermoresponsive polymers solutions, UV and fluorescence detection, microchip-based CE, and entropic trapping networks. 

Y. Baba, M. Tsuhako, Gel-filled capillaries for nucleic acid separations in capillary electrophoresis. Trends Anal. Chem., 1992, n, 280-287. 

J. A. Thompson and coworkers published many excellent papers in the form of both review articles [18-23] and research papers [24-26]. The review articles published in 1986 and 1987 are a series of six publications, each dealing with some aspect of nucleic acid separation. Taken together, the six review articles present a comprehensive description of the different chromatographic methods for nucleic acid separations. 

G. Bonn, C. Huber and P. Oefner, Nucleic acid separation on alkylated nonporous polymer beads, U.S. Pat. 

Thayer, J.R., McCormick, R.M., and Avdalovic, N. (1996) High-resolution nucleic acid separations by high-performance liquid chromatography, in Methods of Enzymology, vol. 271 (eds B. Karger and W.S. Hancock), Academic Press and Elsevier, pp. 147-174. 

---