Nucleic quantitation

Ibrahim and co-workers developed a new method for the quantitative analysis of hypoxanthine, a natural compound of some nucleic acids. " As part of their study they evaluated the method s selectivity for hypoxanthine in the presence of several possible interferents, including ascorbic acid. 

An understanding of a wide variety of phenomena concerning conformational stabilities and molecule-molecule association (protein-protein, protein-ligand, and protein-nucleic acid) requires consideration of solvation effects. In particular, a quantitative assessment of the relative contribution of hydrophobic and electrostatic interactions in macromolecular recognition is a problem of central importance in biology. 

Another property of pyrimidines and purines is their strong absorbance of ultraviolet (UV) light, which is also a consequence of the aromaticity of their heterocyclic ring structures. Figure 11.8 shows characteristic absorption spectra of several of the common bases of nucleic acids—adenine, uracil, cytosine, and guanine—in their nucleotide forms AMP, UMP, CMP, and GMP (see Section 11.4). This property is particularly useful in quantitative and qualitative analysis of nucleotides and nucleic acids. 

James Dewey Watson (1928- ) was born in Chicago, Illinois, and enrolled in the University of Chicago at age 15. He received his Ph.D. in 1950 at the Unwersity of Indiana and then worked at Cambridge University in England from 1951 to 1953, where he and Francis Crick deduced the structure of DNA. After more than 20 years as professor at Harvard University, he moved in 1976 to the Laboratory of Quantitative Biology at Cold Spring Harbor, Long Island, New York. He shared the 1962 Nobel Prize in medicine for his work on nucleic acids. 

Kafil, J. B., Cheng, H.-Y., and Last, T. A., Quantitation of nucleic acids at the picogram level using high-performance liquid chromatography with electrochemical detection, Anal. Chem., 58, 285, 1986. 

BRANCHED DNA SIGNAL AMPLIFICATION FOR DIRECT QUANTITATION OF NUCLEIC ACID SEQUENCES IN CLINICAL SPECIMENS... 

Detecting the presence of a specific nucleic acid sequence may be sufficient for some applications, but there is an increasing demand for quantitation of the... 

The reference standards are used to quantitate the standards that are employed in the kits to generate the standard curves. The kit standards are recombinant single-stranded DNA molecules that are added to either negative serum or plasma at known concentrations. Because the standard curve is not constructed with reference standards, Chiron initially chose to use the term equivalent to describe the units of nucleic acid quantitation in clinical samples. An equivalent was defined as the amount of nucleic acid in a clinical sample that gave a signal equal to one molecule of the reference standard nucleic acid. The term copy rather than equivalent is used to describe the units of nucleic acid quantitation in the HIV-1 bDNA assay. The terms are now used interchangeably. 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

In summary, bDNA has a number of distinct theoretical and practical advantages over target amplification systems for direct quantitation of specific nucleic acid sequences. The following sections review the specific clinical and research applications of this technology. 

The quantitation of HCV RNA in serum may be important in predicting and monitoring response to antiviral therapy (Davis, 1994). A variety of methods have been used to quantitate HCV RNA, including endpoint dilution RT-PCR, competitive PCR, multicyclic RT-PCR, nucleic acid sequence-based amplification, RT-PCR with a single internal quantitation standard, and bDNA (Chazouilleres et al, 1994 Detmer et al., 1996 Ishiyama et al, 1992 Kaneko et al., 1992 Klevits et al., 1991 Kobayashi etal., 1993 Miskovsky etal., 1996). 

Oser, A., Roth, W.K., and Valet, G. (1988) Sensitive non-radioactive dot-blot hybridization using DNA probes labeled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acids Res. 16, 1181-1196. 

Thompson, A., Prescott, M., Chelebi, N., Smith, J., Brown, T., and Schmidt, G. (2007) Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates Synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS. Nucleic Acids Res. 35(4), e28. 

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. 

Didenko, V.V. and Hornsby, P.J. (1996) A quantitative luminescence assay for nonradioactive nucleic acid probes. Journal of the Histochemistry Society, 44 (6), 657-660. 

Skelley et al (1973) listed a number of substances that may be determined quantitatively by the help of the RIA method, namely nucleic acids, proteins, enzymes, prostaglandins, steroidal hormones, antibodies, cancer and viral antigens, vitamins, and drugs together with their respective metabolites. 

Radioactive isotopes provide a very convenient way of monitoring the fate or metabolism of compounds that contain the isotopes. When used in this way, the isotope is described as a tracer and compounds into which the radioactive atom has been introduced are said to be labelled or tagged. The labelled molecules need only comprise a very small proportion of the total amount of the unlabelled radioactive substance because they act in the same way as the non-radioactive substance but can be detected very much more easily. The varied applications of tracers in biochemistry range from studies of metabolism in whole animals or isolated organs to sensitive quantitative analytical techniques, such as radioimmunoassay. Phosphorus-32 is used in work with nucleic acids, particularly in DNA sequencing and hybridization techniques. In these instances the isotope is used as a means of visualizing DNA separations by autoradiographic techniques. 

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