Nucleic acid concentration

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

Equilibrium binding consfanfs can be determined for ligand binding to DNA or RNA, as revealed either from absorbance or fluorescence spectroscopy by keeping a constant concentration of ligand and varying the nucleic acid concentration [125], using the Scatchard equation ... 

A number of animal studies have revealed extrapulmonaiy effects. Again, there is some question as to which of these may represent an effect of ozone, or a direct ozone-induced intermediate, rather than a more indirect response to pulmonary toxicity, perhaps mediated by neurohumoral factors. Thus, for instance, the observations of altered hepatic nucleic acid concentrations, shifts in the content of metals in the liver, alterations in urinary pH, increases in liver weight and alkaline phosphatase... 

ImuVert Manufacturing. The cells were harvested from the fermentation medium and washed by centrifugation, lysed with a French pressure cell and the cell debris was removed by centrifugation. The cell lysis supernatant was then layered on a sucrose gradient and the ImuVert pelleted by centrifugation. The pellet was then resuspended in buffer A (see below) to a nucleic acid concentration of 2 mg/mL. 

Determination of nucleic acid yield. Cleared lysate or ImuVert was diluted to a concentration which had an absorbance between 0.4 and 0.5 at 260 nm and the absorbances at 280 and 260 nm were measured. The nucleic acid concentration in solution was then calculated by the method of Warburg and Christian (18), The yield of nucleic acid was calculated by determining the percent of nucleic acid in the lysate that was isolated in the product. The acceptable range for ImuVert manufacturing is 10.5 + 1.5. 

The high final concentrations of an analyte can result in at least two problems. First, precipitation of nucleic acids can be a problem at high concentrations. The experimentalist should be vigilant during sample preparation to ensure that no signs of precipitation are observed. Second, interactions between nucleic acids can still occur without precipitation. This possibility necessitates controls where BE-AES results are shown to be the same over a range of nucleic acid concentrations. 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

Christel LA, Petersen K, McWilliam W, Northrup MA. Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration. J Biomech Eng 1999 121 272-279. 

Chemical modification of yeast protein has received limited attention though as described above it has potential as a method for facilitating recovery of yeast protein. Current studies are concerned with determination of the functional properties of proteins succinylated during the extraction. The composition of yeast proteins prepared by different methods is shown (Table 8). Noteworthy is the protein and nucleic acid concentration in the yeast isolate which differed from the concentrate in that cell wall material was removed by centrifugation. 

Spectrophotometric methods are most suitable for determining nucleic acid concentrations above about 1 rg ml A solution of double stranded DNA at a concentration of 50 ig ml-1 has an absorbance of 1 at 260 nm, but single stranded nucleic acids have somewhat higher extinction coefficients and a similar absorbance requires only 40 rg ml-1 (ssDNA) and 33 pg ml 1 (ssRNA). It should be noted that these values are mean values for nucleic acids of average base composition. 

Lower nucleic acid concentrations are best determined fluorimetrically. These methods generally depend on the fact that certain dyes can bind to nucleic acids by intercalating between successive base pairs, and this binding is accompanied by marked increases in the fluorescence quantum yield. Ethidium bromide fluorescence (Aex 260-360 nm Af.rr] 560 nm), which is commonly used to visualise nucleic acids in gel electrophoresis, can also be used to quantitate double stranded DNA and RNA with a sensitivity of about 10 ng (Karsten and Wollenberger 1977). The dye 4,6-diamidino-2-phenylindole (DAPI) (Aex 360 nm 2f.m 450 nm) can be used to quantitate DNA specifically with a detection limit of about lng (Brunk et al. 1979). 

Manchester, K.L (1996) Use of UV methods for measurement of protein and nucleic acid concentrations, BioTechniques 20, 968-970. 

DNA and RNA are major N-rich biochemical components of bacteria and viruses, and thus represent important potential sources to the DON pool. Recent methods have found oceanic nucleic acid concentrations in the low pg range (Karl and Bailiff, 1989), suggesting that intact nucleic acids correspond to only a few percent of the total DON pool, despite the fact that rapid turnover suggests they are important N recycling compounds (e.g., Antia et al., 1991 J0rgensen et al., 1993). 

Nucleic Acid Concentration. As the nucleic acid concentration increases, the fraction of covalently bound increases whereas the specific activity of the labeled nucleic acid decreases. Typical data for DNA are shown in Table II. ... 

Figure 1.1. Nomograph used to estimate total protein and nucleic acid concentrations. [Reprinted, with permission, from Calbiochem, San Diego, CA.]...

Rainer TH, Wong LK, Lam W, Yuen E, Lam NYL, Metreweli C> et al. Prognostic use of circulating plasma nucleic acid concentrations in patients with acute stroke. Clin Chem 2003 49 562-9. 

Changes in the rate of synthesis of nucleic acids as measured by the rate of incorporation of labeled precursors may be more informative than changes in nucleic acid concentrations in atrophying muscle. An enhanced incorporation of isotope into nucleic acids after glycine- C is administered to dystrophic mice (aged % to 2 months) was found by Coleman and Ashworth (Cl) the free glycine pool was the same in normal and dystrophic muscle. More extended studies have been reported by Srivastava (S14), who used uridine-2- C as a precursor of RNA. At 30 days of age the incorporation in the dystrophic muscle was higher than normal, but it was the same at 60 days and lower at 90 days. These differences were confirmed by in vitro incorporation experiments... 

The utilization of classical polystyrene particles or hydrophobic latexes for protein concentrations can induce undesirable phenomena such as protein denaturation and low concentration yields, on account of the high adsorption affinity between both species which may lead to a low desorbed amount. In addition, the use of such hydrophobic colloids in the polymerase chain reaction (PCR) of nucleic acid amplification step generally leads to total inhibition of the enzymatic reaction. The inhibition phenomena can be attributed to the denaturation of enzymes adsorbed in large numbers onto hydrophobic coUoids. The utilization of hydrophilic and highly hydrated latex particles (irrespective of temperature) is the key to solving this problem by suppressing the inhibition of enzyme activity. The purpose of this stage is then to focus on the potential apphcation of thermally responsive poly(NIPAM) particles for both protein and nucleic acid concentrations. 

II. Influence of Vitamin E Deficiency on Tissue Nucleic Acid Concentrations. 512... 

Our own experiments, which were started approximately ten years ago, have utilized rats, rabbits, and monkeys, and we have investigated the influence of vitamin E deficiency on tissue nucleic acid concentrations in... 

Nucleic acid concentrations are expressed as mtlligranis nudeic add P per gram wet weight of tissue. 

The data in Table I summarize some of the findings on the influence of vitamin E deficiency on tissue nucleic acid concentrations. In the monkey and rabbit, vitamin E deficiency results in a great increase in the concentration of DNA per gram wet weight of skeletal muscle. This is in agreement with the histologic picture of these tissues, which is characterized by an increase in number of sarcolerama nuclei. Also in the monkey there is a considerable increase in the concentration of DNA and RNA in bone marrow. 

UV absorption spectroscopy is further used to determine nucleic acid concentration. The average molecular weight of a nucleotide is 320 and 50[rgml concentration of a random sequence DNA provides the A260 absorbance of 1 in a 1 cm pathlength cell. In practice, the absorption coefficient of DNA is precisely determined by enzymatic hydrolysis of DNA to monomers and calculation using the absorption coefficients of the monomers, or by measurement of thermally denatured DNA. The absorption of the denatured DNA sample is calculated on the basis of absorption coefficients of the constituent mono- and dinucleotides. 

Foster AR, Houlihan DF, Gray C, Medale F, Fauconneau B, Kaushik SJ, LeBail PY (1990a) The effects of ovine growth hormone on protein turnover in rainbow trout. Gen Comp Endoain (In Press) Foster AR, Houlihan DF, Hall SJ (1990b) The effect of temperature on protein synthesis and nucleic acid concentration/ratios in juvenile cod. (in preparation)... 

In the sample preparation step, the nucleic acid content from the sample is extracted, concentrated, and purified to meet the quality required by the downstream amplification process. Sample preparation of nucleic acid targeting technologies involves several processes. In most cases, lysis is an initially required process to break open intact structures of targeted species (e.g., cell wall, membrane, and capsules) and to release nucleic acids. Additional purification or extraction of the released nucleic acids from the lysate may be conducted to minimize inhibitory effects on downstream amplification. Following the lysis step, the chemicals introduced (e.g., membrane-solubilizing ionic detergents or proteinases), cell debris, and other interferents that come with samples are removed. For diluted specimens (i.e., urine), nucleic acid concentration may be required in order to reduce the limit of detection of amplification and detection assays. 

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