Nucleic acids separation techniques

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. 

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. 

Techniques for separation of DNA and RNA provide powerful tools for the analysis of genetic material and transcription products. Modem methods of analysis can answer most of the questions about the sequence, nucleotide substitutions (mutations, polymorphisms, or single nucleotide polymorphisms -SNPs), and stmcture of nucleic acids. Crucial techniques in the analysis of DNA or RNA are the... 

Centrifugation can be used either as a preparative technique for separating and purifying macromolecules and cellular components or as an analytical technique to characterize the hydrodynamic properties of macromolecules such as proteins and nucleic acids. 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

As discussed in Chapter 11, electrophoresis refers to a group of techniques used to separate and study molecules with electrical charges. Based upon these charges, both sign and magnitude, biomolecules such as proteins, peptides, amino acids, nucleic acids, and fragmented nucleic acids migrate in an electrical... 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

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