Quantification of DNA

Unfortunately these and other existing quality control procedures do not answer aU problems. There remains a clear need for development of PCR reference materials that win provide information both on quality and quantity levels. For quality the reference materials should be host-specific and PCR primers, for positive control, may correspond to host specific house keeping genes e.g. b-actin. For quantitative analysis, fluorescence dyes in specific primers might be used in order to measure accurately the amount of DNA present. Such practices, and other as yet un-realized procedures, will be needed to achieve reliable results in the quantification of DNA analysis. 

Mohn, G.R., Kerklaan, P.R.M.. Van Zeeland, A. A., Ellcnberger, J., Baan, R.A., Lohman, P.HM. Pons, F.W. (1984) Methodologies for the determination of various genetic effects in permeable strains of E. coU K-12 differing in DNA repair capacity. Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal-mediated assays. Mutat. Res., 125, 15,3-184... 

UV spectroscopic methods were used to determine equilibrium constants between cyanine dyes (thiazole orange) and nucleic acid <2005JA3339, 2002PCB4838,1999ABI278>, quantification of DNA <1998AEM3238>, and Ru(n), and Ni(ll) complexes containing thiazoles <2003IC8038>. 

Quantification of DNA adducts in samples at very low exposure levels, and after administration of very low quantities of isotopes/ radiolabeled compounds (as low as lpCi/40kBq). 

A probe that can detect all target DNA (such as a vector probe) will allow the relative quantification of DNA in each spot to be determined. Expression levels can then be compared across several slides after the inevitable differences in quantity of target DNA binding to the slide have been taken into account. This approach can only be taken if all target DNA samples are tagged with the same DNA element. 

E., and Dedon, P.C. (2008) Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation hy liquid chromatography isotope dilution tandem mass spectrometry. Nat. Protoc., 3,1287-1298. 

We used BART for quantification of DNA in two different iNAATs Lamp-Mediated Amplification (LAMP)2 and chimeric primers amplification (RDC)3. Both amplifications show sensitivity down to 10 copies of DNA and the BART reagent has no adverse effect on the sensitivity. Good linear correlation between Peak time and DNA copy number is observed in both cases independent of the length of the assay (Fig. 4c-f). BART reagent retained high activity for 4 hours at 60°C. 

Fig. 2. BART quantification of DNA in the absence (black line) and presence (grey line) of high background carrier DNA.

Quantification of DNA Spectrophotometry and fluorometry are commonly used to measure DNA concentration. Spectrophotometry can be used to measure microgram quantities of pure DNA samples (i.e., DNA that is not contaminated by proteins, phenol, agarose, or RNA). Fluorometry is more sensitive, allowing the measurement of nanogram quantities of DNA, furthermore the use of Hoedist 33258 dye or picogreen allows more specific analysis of DNA... 

Fluorometry Fluorometry allows specific and sensitive measurement of DNA concentration by the use of the fluorochrome Hoechst 33258, which shows increased emission at 458 nm when bond to DNA. This dye has little affinity for RNA, allowing accurate quantification of DNA samples that are contaminated with RNA. 

Characterization of these antibodies was carried out by competitive ELKA. The difference in lA g values between 60 and 2.4 adducts per 10 nucleotides was in the order of 30. Namely, the binding reaction was 30 fold greater with the highly modified DNA. Thus for quantification of DNA adducts of this type in biological samples, DNA with a low degree of modification must be used as the competitor standard.. ... 

Song B, Vandevyver CDB, Deiters E, Chauvin AS, Henunila I, Btinzli JCG (2008) A versatile method for quantification of DNA and PCR products based on time-resolved Eu luminescence. Analyst 133 1749-1756... 

HoeUcer M, Mekchay S, Schneider H et al (2007) Quantification of DNA binding, uptake, transmission and expression in bovine sperm mediated gene transfer by RT-PCR effect of transfection reagent and DNA architecture. Theriogenology 67 1097-1107... 

Henry OY, Acero Sanchez JL, Latta D, O Sullivan CK (2009) Electrochemical quantification of DNA amplicons via the detectirai of non-hybridised guanine bases on low-density electrode arrays. Biosens Bioelectron 24 2064-2070... 

Harriman, W. D. Wabl, M. A video technique for the quantification of DNA in gels stained with ethidium bromide. Anal. Biochem. 1995, 228, 336-342. 

---