End-labelling method

This method is useful for small RNAs and complements the primer extension method at the 3 end of the RNA where information is lost due to primer annealing. The method requires RNA with at least one homogeneous end. Furthermore, strand scission is re- 

Care should be taken in the assignment of the modified residues. This is most conveniently done by co-electrophoresis of a sequencing track. However, as illustrated in Fig. 4.12 the termini generated by the different modifications and sequencing procedures vary so the fragments may not co-migrate. 

The enzymatic sequencing procedure often referred to as the Donis- 

Keller method10 is based on a partial digestion of end-labelled 

RNA with nucleotide-specific RNases. The digests produce G, A, A + U and U + C lanes. Three reactions are performed under denaturing conditions in the presence of urea that ensures a uniform band intensity independent of the secondary structure. The U + C reaction is performed in the absence of denaturing agents and consequently the band intensities depend on the secondary structure. 

---

Itoh G, Tamura J, Suzuki M, Suzuki Y, Ikeda H, Koike M, et al. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 1995 146 1325-1331. 

There are two different methods to identify modified residues and ribonuclease scissions in RNA molecules the reverse transcriptase method or the end-labelling method. The choice of method depends both on the length of the studied RNA and the method of probing (as discussed in Sections 4.4.1 and 4.4.2). The reverse transcription method uses extension of a primer and therefore is independent of the length of the RNA, while the end-labelling method is restricted to analysis of small RNA molecules (n < 300). The latter method requires scission of the RNA. 

Modified residues can be detected by reverse transcriptase methods or end-labelled methods after an acidic aniline cleavage (Section 4.4.3). Hydrazine modifications can only be performed under denaturing conditions. 

Maxam, A. M. Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980, 65, 499-560. 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

Transfer RNA (tRNA) molecules mediate translation of the nucleic acid genetic code into the amino acid building blocks of proteins, thus ensuring the survivability of cells. The dynamic properties of tRNA molecules are crucial to their functions in both activity and specificity. This chapter summarizes two methods that have been recently developed or improved upon previous protocols to introduce fluorophores to site-specific positions in tRNA. One method enables incorporation of fluorophores carrying a primary amine (such as proflavin or rhodamine) to dihydrouridine (D) residues in the tRNA tertiary core, and a second method enables incorporation of pyrroloC and 2-aminopurine to positions 75 and 76, respectively, of the CCA sequence at the 3 end. These site-specific fluorophore labeling methods utilize tRNA transcripts as the... 

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

Biochemically, apoptosis is characterized by the internucleosomal degradation of chromosomal DNA to form a series of double-stranded fragments that are multiples of 180 200 base pairs in length. These fragments give a characteristic DNA ladder pattern on gel electrophoresis [91, 92] and can be detected by several cytochemical methods, the most extensively used being the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) [93-95], The detection of ladder pattern and TUNEL positivity has been adopted as a marker of apoptosis. 

Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH. A new method to detect apoptosis in paraffin sections In situ end-labeling of fragmented DNA. J Histochem Cytochem 1993 41 7-12. 

Detailed procedures for 5 - or 3 -end labelling DNA fragments, following the method of Maxam and Gilbert (1980) are given in Chapter 5. Essentially similar protocols are given by Wu et al. (1976) and Roychoudhury and Wu (1980). 

The principle of the method is shown in Figure 3.9. It might be thought that the 5 - 3 exonuclease activity of the DNA polymerase would result in the removal of 5 -end label from the DNA fragment. However, this does not seem to be a serious problem and experience (Maat and Smith, 1978) has shown that there is no significant loss of labelled material due to hydrolysis of the labelled 5 -end. 

---