Cytosine detection

Figure 3.12 depicts TOP SIMS spectra obtained from ODN and PNA immobilized on silanized silicon wafers. The spectra clearly demonstrate that the masses corresponding to POi and PO3 provide the best correlation of the presence of ODN, enabling their use for precise distinction between ODN and PNA. The CFJ and C2O2FJ peaks seen in the PNA spectra represent trifluoroacetic acid, which was part of the PNA solution. Deprotonated (Cyt-H) and (Thy-H) signals of the bases cytosine and thymine are observed for both immobilized PNA and ODN sequences and can be used to detect the presence of these bases. 

Uracil, thymine, and cytosine have been studied using this technique (89JA2308 and references therein). For uracil and thymine, the dioxo tautomer predominates in the case of cytosine (70), three tautomers were detected, 70a, 70b, and 70c, the last one being the least abundant. The gas-phase tautomeric equilibrium of 2-pyridone 15a and 2-hydroxypyridine 15b has been studied by MW spectroscopy (93JPC46) using both a conventional spectrometer and a jet-cooled millimeter-wave spectrometer. The relative abundances are 3 1 in favor of the hydroxy form 15b, which exists in the Z conformation shown (Scheme 23). 

Kinoshita, Imoto etal.11 14) synthesized other anionic models, 5 (APVP), CPVP, UPVP, TPVA, HPVA, THPVA, and 6 (AMPPVA), by the polymer reaction of N-eoupled(2-dihydrogenphosphate)-ethylderivatives of nucleic acid bases (or adenosine-5 -phosphate, AMP) with polyvinylaleohol. A, C, U, T, H, and TH denote adenine, cytosine, uracil, thymin, hypoxanthine, and theophylline, respectively. The authors reported the apparent hypochromities of 3 to 16% for many kinds of mixtures of the models and DNA or RNA, as compiled in Table 1. However, for the mixtures APVA + RNA, HPVA + RNA HPVA + DNA, THPVA + RNA, CPVA + DNA and CPVA + RNA, no hypochromicity was detected. 

Figure 12 Gradient separation of bases, nucleosides and nucleoside mono- and polyphosphates. Column 0.6 x 45 cm. Aminex A-14 (20 3 p) in the chloride form. Eluent 0.1 M 2-methyl-2-amino-l-propanol delivered in a gradient from pH 9.9-100 mM NaCl to pH 10.0-400 mM NaCl. Flow rate 100 ml/hr. Temperature 55°C. Detection UV at 254 nm. Abbreviations (Cyt) cytosine, (Cyd) cytidine, (Ado) adenosine, (Urd) uridine, (Thyd) thymidine, (Ura) uracil, (CMP) cytidine monophosphate, (Gua) guanine, (Guo) guanosine, (Xan) xanthine, (Hyp) hypoxanthine, (Ino) inosine, (Ade) adenosine, (UMP) uridine monophosphate, (CDP) cytidine diphosphate, (AMP) adenosine monophosphate, (GMP) guanosine monophosphate, (IMP) inosine monophosphate, (CTP) cytidine triphosphate, (ADP) adenosine diphosphate, (UDP) uridine monophosphate, (GDP) guanosine diphosphate, (UTP) uridine triphosphate, (ATP) adenosine triphosphate, (GTP), guanosine triphosphate. (Reproduced with permission of Elsevier Science from Floridi, A., Palmerini, C. A., and Fini, C., /. Chromatogr., 138, 203, 1977.)...

Robertson et al. (1996) were successful in synthesising cytosine and uracil in amazingly high yields. They started from cyanoacetaldehyde (obtained via hydrolysis of cyanoacetylene) and, again, urea. Cytosine is not formed in detectable concentrations unless concentrated urea solutions are used then, however, the yields are between 30 and 50%. Uracil was obtained by hydrolysis of cytosine solutions. 

Hydantoin, the cyclic form of hydantoic acid, was detected in the Murchison meteorite and also as a product of polymerisation of HCN (Ferris et al 1974). The yields obtained (based on cyanoacetaldehyde) were 18% for cytosine-N -acetic acid, but only 1.8% for the corresponding uracil derivative (1 mM cyanoacetaldehyde and 2 M hydantoic acid were allowed to react at around 373 K). 

Bisulfite modification of cytosine residues also can be used to add permanently a sulfone group to the C-6 position. In this scheme, the sulfone functions as a hapten recognizable by specific anti-sulfone antibodies. At high concentrations of bisulfite and in the presence of methyl-hydroxylamine, cytosines are transformed into N4-methoxy-5,6-dihydrocytosine-6-sulphonate derivatives (Herzberg, 1984 Nur et al., 1989). Labeled antibodies can then be used to detect the hybridization of such probes. 

Also for cytosine, red shifts are detected in the NH2 stretching and bending modes. Ring stretchings and deformations, involving the CO vibration, suffer little changes with respect to the isolated base (see Fig. 16). In the overall, the computed frequency shifts are in agreement with the... 

Although the hybridization of single-stranded DNA to its complement results in detectable changes in electrochemical properties, particularly in support of non-Faradaic current, the DNA bases may also demonstrate redox behavior that gives rise to Faradaic currents. The electrochemical behavior of DNA has been studied over the past few decades. Differential pulse voltammograms show clearly defined peaks for the reduction of cytosine and adenosine. Electrochemical characterization of guanine by cyclic voltammetry has shown... 

ChIP-chop allows one to determine if the DNA that is associated with an antigen of interest is highly CpG-methylated. The Escherichia coli enzyme McrBC is used to hydrolyze ( chop ) the DNA that is methylated specifically at purine/cytosine sites. Hydrolysis within the target DNA sequence prevents detection of the DNA by PCR [24]. 

L.D. Stegman, A. Rehemtulla, D.A. Hamstra, D.J. Rice, S.J. Jonas, K.L. Stout, T.L. Chenevert, B.D. Ross, Diffusion MRI detects early events In the response of a glioma model to the yeast cytosine deaminase gene therapy strategy. Gene Then 7 (2000) 1005-1010. 

The most important pyrimidine derivatives are those upon which biological organisms depend. Cytosine 1018 and uracil 1019 are found in ribonucleic acid (RNA) in the form of their ribonucleotides, cytidine 1020 and uridine 1021, while in deoxyribonucleic acid (DNA), cytosine and thymine 1022 are found in the form of their 2 -deoxyribonucleotides, 2 -deoxycytidine 1023 and thymidine 1024. 5-Methylcytosine 1025 is also found to a small extent (c. 5%) in human DNA in the form of its 2 -deoxyriboside 1026, and 5-(hydroxymethyl)cytosine-2 -deoxyriboside 1027 has also been detected in smaller amounts <2005CBI1>. Many variants of cytosine and uracil can be found in RNA including orotic acid 1028 in the form of its ribonucleotide orotidine 1029. Other pyrimidine derivatives to have been isolated from various biological sources include 2 -deoxyuridine 1030, alloxan 1031, and toxopyrimidine (pyramine) 1032 (Figure 2). 

The existence of photoreversible, but not of heat-reversible, absorbance change in irradiated poly dI dC was taken to prove that the photoproducts are entirely dimers (in contrast to those in poly C irradiations where the product is almost entirely the hydrate82a). It was possible to detect dimers of uracil as well as those of cytosine, by means of the much slower photoreversal of uracil dimers. In the acid hydrolysates of irradiated dl-dC, both uracil dimers and uracil could be identified. Enzymatic hydrolysis (snake venom phosphodiesterase) does not split pyrimidine dimers, and the products of such hydrolysis of irradiated tritium-labeled poly dl dC contained trinucleotides shown by radioactivity to contain cytosine dimers. Thymine dimers were formed in the photolysis of the poly dA dT, and were detected and assayed by the same methods. The yield of thymine dimers in irradiated poly... 

Levin, D.E., Yamasaki, E. Ames, B.N. (1982) A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A nm of cytosines as a mutational hot-spot. Mutat. Res., 94, 315-330... 

A common source of DNA damage is the spontaneous loss of the amine group on cytosine and the formation of an amide. This occurs at a rate of about 100 times a day. Fortunately, the body produces enzymes able to detect and repair such degraded cytosines. Given this information, suggest why DNA differs from RNA in possessing the nucleotide thymine rather than uracil. 

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