Nucleotide Labeling Methods

In the direct labeling method, mRNA is converted into a labeled cDNA population. This is achieved by copying the transcripts into cDNA molecules with a reverse transcriptase while incorporating a modified Cy dye nucleotide. 

In the indirect labeling method, amine modified cDNA is first synthesised by incorporating aminoallyl-modified nucleotides in first strand cDNA by a reverse transcriptase. After hydrolysis of the RNA template, and purification of the amine-modified cDNA, chemical labeling with N-hydroxyl succinimidyl-ester derivative of the Cy dye is performed. A high excess of Cy dye NHS-ester is needed for an efficient reaction. The Cy dye-cDNA is then purified to remove Cy dye that is not incorporated into labeled cDNA. 

A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [112]. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114,115]. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. 

The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. 

Radio- iso- tope Half- life Radiation type/max energy of emission (MeV) (mean) Typical SA of nucleotides (Ci/mmol) Labeling methods Typical SA of probe (dpm/p.g) Probe detection limit (pg/cm )... 

Fuller, C. Kumar, S. Sood, A. Nelson, J. Xerminal-phosphate-labeled nucleotides and methods of use. U.S. Pat. Appl. Publ. US 20030162213,2003. 

Houthoff, H. J. Reedijk, J. Jelsma, T. Heetebrij, R. J. Volkers, H. H. Methods for labeling nucleotides, labeled nucleotides and useful intermediates. Application Information W0 977NL559 19971008... 

To modify the unique chemical groups on nucleic acids, novel methods have been developed that allow derivatization through discrete sites on the available bases, sugars, or phosphate groups (see Chapter 1, Section 3 for a discussion of RNA and DNA structure). These chemical methods can be used to add a functional group or a label to an individual nucleotide or to one or more sites in oligonucleotide probes or full-sized DNA or RNA polymers. 

Before using the probe for hybridization, it must be labelled to enable subsequent detection. One method of labelling is by the incorporation of 32P-labelled nucleotides, a process that may be carried out in two ways. 

Figure 13.19 The Maxam and Gilbert method of DNA sequencing. The labelled DNA strand is divided into four aliquots. Each is treated to cleave the strand next to a different base resulting in a mixture of different length nucleotides. These nucleotides are separated by polyacrylamide gel electrophoresis and the DNA sequence read from the gel.

---