Cytosine labeling with

Ultraviolet light photolysis of CpC (16c) leads to the formation of mono- and dihydrates and dimers of the cytosine residue. Since saturation of the 5,6-double bond labilizes the 4-amino group, deamination also takes place readily with the formation of hydrates and dimers also of UpC, CpU, and UpU. A chart of those products which have been separated and identified is shown in Figure 21.74 The starting material, CpC, was labeled with 32P. The products were separated by various... 

The borohydride reduction of 2 - and 3 -ketocytidine derivatives having or lacking an N4-acetyl group provided a facile route to cytosine nucleosides labeled with tritium at specific positions of the sugar.28 Reduction of either 20a or 20c with sodium borohydride in ethanol-benzene afforded the arabino and the ribo derivatives in the ratio of 4 1. Treatment of the reduction mixture with methanolic ammonium hydroxide gave the deacetylation products, 82b and 19b, isolated in crystalline form by preparative, thin-layer chromatography. 

Nucleic acids can be labeled with a radioactive isotope in vitro by heating them in a mixture containing radioactive iodine and thallic trichloride (TICI3). The reaction is rapid and simple. It results in the formation of a stable covalent bond between the radioactive iodine atom and carbon atom 5 of cytosine in the nucleic acid. The biological properties of the nucleic acid are not significantly affected by this procedure. 

Find the label. Suppose that cells are grown on amino acids that have all been labeled at the ct carbons with C. Identify the atoms in cytosine and guanine that will be labeled with C. 

Ring carbons 4, 5, and 6 in cytosine will be labeled. In guanine, only the bridge carbon atoms that are shared between the 5- and 6-membered rings will be labeled with C. 

In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

Deoxycytidine (dCyd) (14 in Scheme 2) is also an excellent target for one-electron oxidation reactions mediated by triplet excited menadione. On the basis of extensive identification of dCyd photooxidation products, it was concluded that this nucleoside decomposes by competitive hydration and deprotonation reactions of cytosine radical cations with yields of 52% and 40%, respectively [53]. It was also found, on the basis of 180 labeling experiments, that hydration of cytosine radical cations (15) predominantly occurs... 

An analogous series of reactions is used to produce depyrimidinated DNA fragments. Hydrazine is used in these reactions, since both cytosine and thymine react with hydrazine. The bases are cleaved to yield urea and a pyrazole ring. The deoxyribose moiety is left as a hydrazone. Piperidine, which reacts with the hydrazone, is used to cleave the nucleotide chain. Cytosines react specifically with hydrazine in 5 mol/ L NaCl, but no specific reaction exists for thymines. Consequently, one aliquot yields labeled oligonu-cleotides 3 -terminated at cytosines, whereas a second aliquot contains nucleotides cleaved in the absence of NaCI at both cytosine and thymine residues. 

The hydrazide derivative of AMCA can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure described in Chapter 27, Section 2.1. AMCA-hydrazide reacts with these target groups to form hydrazone bonds (Figure 9.26). Carbohydrates and glycoconjugates can be labeled specifically at their polysaccharide portion if the required aldehydes are first formed by periodate oxidation or another such method (Chapter 1, Section 4.4). 

Color Plate 29 DNA Sequencing by Capillary Gel Electrophoresis with Fluorescent Labels (Section 26-6) Tall red peaks correspond to chains terminating in cytosine and short red peaks correspond to thymine. Tall blue peaks arise from adenine and short blue peaks indicate guanine. Two different fluorescent labels and two fluorescence wavelengths were required to generate this information. [From M. C. Ruiz-Martinez, J. Berka, A. Belenkii,... 

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

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