In DNA isolation

Fig. 22. Numbers of intrastrand crosslinks to GG-units in DNA isolated from white blood cells of two different patients at various time intervals after the onset of the cisplatin infusion (indicated by arrows).

Once purified, DNA is a fairly stable polymer if stored appropriately. Since living cells contain many other complex biomolecules besides DNA, methods exist that allow the isolation of DNA in pure form. More details on this topic are presented in Chapter 8. Routine methods of DNA isolation in solution, however, cause some unavoidable shearing of DNA due to hydro-dynamic shear forces, and as a result, the average size of isolated DNA is about 100 to 200 kilobases (kb). The basic steps in DNA isolation involve cell disruption and lysis by treatment with detergents, removal of cellular proteins by either enzymatic digestion with a protease or extraction with... 

The determination of the total platinum content in physiological fluids and tissues, both during clinical treatment or after environmental exposure, requires instrumental techniques of sufficient DLs and selectivities. ICP MS provides the most attractive DLs for platinum in biological samples, for example, 7.50 ng/L in human plasma ultrafiltrate after chemotherapy with cisplatin, carboplatin, and oxaliplatin [119] 0.1 pg/mL in blood, serum, and ultrafiltrate samples after chemotherapy with oxaliplatin and 5-fluorouracyl [120] 26 pg/g in DNA isolated from cancer ovarian cells after different exposure times and concentrations of cisplatin [121] and 0.75 pg in DNA extracts from peripheral blood mononuclear cells and tissues from patients treated with cisplatin [122] and 1.0 pg/L in serum, 0.1 pg/L in ultrafiltrate, and 2 pg/L in urine [123]. The ICP MS technique allowed detection of physiological levels of ft in the tmexposed human body 0.3-1.3 ng/L in blood (DL of 0.3 ng/L) [46] 0.48-7.7 ng/L in urine (DL of 0.24 ng/L) [47] and 0.778 ng/g... 

Shukla LI, Pazdro R, Becker D, SeviUa MD. (2005) Sugar radicals in DNA Isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging. Radiat Res 167 501-507. 

The role of depurinating adducts and apurinic sites in the PAH-induced cancer process is controversial and has yet to be fully elucidated. There are lines of evidence that both support and refute this theory. In support of this theory, the levels of depurinating adducts of B[a]P correlated with mutations in the H-ras oncogene in DNA isolated from mouse skin papillomas initiated by this compound (Chakravarti et al. 1995). It is well known that the initiation of skin tumors in mice is associated with the formation of mutations in the H-ras gene [reviewed by Ross and Nesnow (1999)]. DB[a,/]P treatment of mouse skin forms papillomas which contain the H-ras codon 61 (CAA to CTA) mutation. These same mutations were induced in early preneoplastic skin within one day after DB[a,/]P treatment and appear to be related to DB[a,/]P-Ade-depurinating adducts. Studies have shown that apurinic... 

Peroxyl radicals produced strand breaks and base modification in DNA isolated from human male fibroblasts grown in 150 mm dishes to confluent monolayers (Rodriguez et al. 1999). Oxidative base modifications were observed to occur at a greater extent than strand breaks at every concentration measured. A total of 87% of all guanine... 

Many cells that comprise the vasculature generate reactive oxygen (for review see Irani 2000) and/ or nitrogen species. The amount of 8-hydroxy-2 -deoxyguanosine, one of the typical biomarkers of oxidative stress, in DNA isolated from lymphocytes of 43 atherosclerotic patients with intermittent... 

The DNA isolated from different cells and viruses characteristically consists of two polynucleotide strands wound together to form a long, slender, helical molecule, the DNA double helix. The strands run in opposite directions that is, they are antiparallel and are held together in the double helical structure through interchain hydrogen bonds (Eigure 11.19). These H bonds pair the bases of nucleotides in one chain to complementary bases in the other, a phenomenon called base pairing. 

The consequence of ADA deficiency is accumulation of adenosine and 2 -deoxyadenosine, substances toxic to lymphocytes, important cells in the immune response. 2 -Deoxyadenosine is particularly toxic because its presence leads to accumulation of its nucleotide form, dATP, an essential substrate in DNA synthesis. Elevated levels of dATP actually block DNA replication and cell division by inhibiting synthesis of the other deoxynncleoside 5 -triphosphates (see Chapter 27). Accumulation of dATP also leads to selective depletion of cellular ATP, robbing cells of energy. Children with ADA SCID fail to develop normal immune responses and are susceptible to fatal infections, unless kept in protective isolation. 

Samples of DNA isolated from different tissues of the same species have the same proportions of heterocyclic bases, but samples from different species often have greatly different proportions of bases. Human DNA, for example, contains about 30% each of adenine and thymine and about 20% each of guanine and cytosine. The bacterium Clostridium perfringens, however, contains about 37% each of adenine and thymine and only 13% each of guanine and cytosine. Note that in both examples the bases occur in pairs. Adenine and thymine are present in equal amounts, as are cytosine and guanine. Why ... 

In another series of experiments, a novel approach to the determination of nucleotide sequence was adopted by A. S. Jones, Stacey, and their co-workers. For example, when calf thymus DNA was treated with mercaptoacetic acid in the presence of zinc chloride and anhydrous sodium sulfate, it yielded aldehydo-apurinic acid bis(carboxymethyl) dithioacetal. When degraded with dilute alkali, this afforded dialyzable fragments, which were separated into at least 20 components. Some were identified, including mono-, di-, and tri-nucleotides, thereby revealing that DNA contain regions of at least three linked pyrimidine nucleotides. The same procedure was applied to the DNA isolated from M. phlei ... 

Figure 3.9 Cloning and DNA-isolation strategies commonly used in metagenomics.

When the histone octamer is mixed with purified, double-stranded DNA, the same x-ray diffraction pattern is formed as that observed in freshly isolated chromatin. Electron microscopic studies confirm the existence of reconstituted nucleosomes. Furthermore, the reconsti-mtion of nucleosomes from DNA and histones H2A, H2B, H3, and H4 is independent of the organismal or cellular origin of the various components. The histone HI and the nonhistone proteins are not necessary for the reconstitution of the nucleosome core. 

C18-0120. You are doing undergraduate research for a biology professor. Your first assignment is to prepare a pH =7.50 phosphate buffer solution to be used in the isolation of DNA from a cell culture. The buffer must have a total concentration of 0.500 M. On the shelf you find the following chemicals solid NaOH concentrated HCl (12.0 M) concentrated H3 PO4 (14.7 M) KH2 PO4 and K2 HPO4. Write a quantitative detailed set of instmctions that describe how you would prepare 1.5 L of the buffer solution. 

Since the results of our experiments with isolated rat liver fractions supported a reaction sequence Initiated by microsomal oxidation of the nitrosamine leading to formation of a carbonium ion, the results of the animal experiment suggested that in the intact hepatocyte, one of the earlier electrophilic intermediates (II, III or V, Figure 1) is intercepted by nucleophilic sites in DNA (exemplified here by the N7 position of guanine) before a carbocation is formed. 

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