Nucleotides, labeled elution

Fig. 8.7 The principle of full genome sequencing using labelled dideoxy (dd) nucleotides labelled fragments are eluted through a gel and detected in order of length to determine the complementary DNA sequence from which the original can be deduced...

After labelling ragged ends cleave with a restriction enzyme to yield end-labelled fragments of less than 100 nucleotides. Denature and electrophorese on a sequencing gel (1.5 mm thick). Select a well-resolved single band, elute, and use for sequence analysis... 

Fig. 2.1. Nucleotide fractionation. A sample of p2 phage RNA uniformly labelled with and the pyrimidines labelled with was digested with alkali ( 2.1.1) and separated by column chromatography on Dowex 50 formate eluted with 0.25 M ammonium formate pH 4.1. The ultraviolet transmission graph is traced from the record of a Uvicord I ultraviolet monitor output Radioactivities were measured by dissolving the samples in a scintillation cocktail and counting in a liquid scintillation counter. The peaks were identified from their ultraviolet spectra.

Elute labeled probe m 100 pL of water using a nucleotide removal column (see Note 11). 

An earlier variation of the elution method was described in a detailed study by Ives et al., (1969). Using 0.1 M HCl -0.2 M KCl eluant with H labelled nucleotides they successfully estimated the activity of some ribo-, and deoxyribonucleoside kinases and even that of various enzymes which hydrolyze nu-... 

In conclusion, our results showed that counting labeled nucleotides directly on dried DEAE disks produces erroneous results. The failure to recognize this is responsible for the variable and low thymidine kinase activities in the cerebellum reported in previous studies (Weichsel, 1974, Weichsel and Dawson, 1976). The elution of the labeled nucleotides with 1 ml of 1 M HCl containing 0.5 M NaCl in the counting vial in improved counting efficiency and reproducibility. Furthermore, it is not necessary to dry the disks, thus the overall method became less time-consuming. 

Separation of RNase T2 digests of 52p-labeled polio virion RNA (a) mRNA (b) and replicative intermediate ENA (c) on DEAE-cellulose in the presence of marker nucleotides. Columns were in 5 i al plastic pipettes (Falcon, Serological) elution with triethylammonium acetate, pH 5, was as described (5, 111 12). 

Fig. 1 shows such a separation in a PGA extract of H-Uridine labelled erythrocytes from a patient with pyrimidine 5 nucleotidase deficiency. Peaks corresponding in elution time to uridine and cytidine mono-, di-, and tri-phosphates can be seen. Label is present in all three uridine nucleotide peaks as well as in a fourth peak which corresponds in elution time in these and under other separation conditions with UDP-glucose (UDPG). 

Rabbit livers, perfused free of blood, were then labeled by perfusion, in situ, via the portal vein, with a suitable purine nucleotide precursor tritium labeled adenine or hypoxanthine. The livers were then perfused, with an isotonic solution containing a carrier nucleoside, usually unlabeled adenosine Hepatic venous effluent was collected, during five minute periods, for twenty minutes. Cold dilute perchloric acid extracts were prepared and the purine components eluted by ion exchange chromatography on Dowex 50. The data obtained, following labeled adenine perfusion and subsequent perfusion with unlabeled adenosine are presented in Figure 1. 

---