Nucleoside triphosphates labeled

In this experiment, you will carry out an in vitro transcription reaction from a plasmid (pSP72, Fig. 22-4) containing a T7 RNA polymerase specific promoter. A 32P-labeled nucleoside triphosphate (labeled at the a position) will be included in the reaction to produce a radioactive RNA molecule. The size and sequence of the various transcripts that you will produce can be predicted by the restriction enzymes that the plasmid will be digested with prior to the beginning of the transcription reaction. Since the plasmid template for the transcription reaction will be digested, you will be preparing runoff transcripts from the T7 promoter. As a result,... 

Nick translation A technique for labeling DNA based on the ability of the DNA polymerase from E colt to degrade a strand of DNA that has been nicked and then to resynthesize the strand if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe. 

Terminal transferase labeling was originally developed using radiolabeled (typically 32P) nucleoside triphosphates (Roychoudhury et al., 1979 Tu and Cohen, 1980). Later, the technique was extended to the use of nonradioactive nucleotide derivatives (Kumar et al., 1988). 

Regardless of the type of enzymatic labeling used, it is important that the label be incorporated into the nucleoside triphosphates or primers in a way that does not affect enzyme recognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA probes must start with chemical derivatization of individual nucleotides. Of the many chemical procedures that can be used to modify a nucleoside triphosphate monomer, there are only a few that will result in a derivative still able to be enzymatically added to an existing oligonucleotide strand. 

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Some form of chemical labeling process must be used regardless of whether the final oligo conjugate is created by enzymatic or strictly chemical means. If enzymatic modification is to be done, the initial label still must be incorporated into an individual nucleoside triphosphate, which then is polymerized into an existing oligonucleotide strand (Section 1, this chapter). Fortunately, many useful modified nucleoside triphosphates are now available from commercial sources, often eliminating the need for custom derivatization of individual nucleotides. 

Separation and Quantitation.—The specific radioactivity of [y-32P]ATP of very high activity (up to 240 Ci per millimole) may be measured by using it to phosphorylate [dT(pT)10] quantitatively, using polynucleotide kinase, isolating the labelled undecanucleotide, and measuring its activity.170 Isotope dilution is used to confirm the values. An alternative method of measuring specific radioactivities of ribo-nucleoside triphosphates utilizes a 3H-labelled nucleoside triphosphate (e.g. UTP) of unknown specific activity, a 14C-labelled nucleoside (e.g. ATP), a suitable primer in... 

The solution structures of 1 1 complex between paromomycin and gentamicin Cla and the A-site oligonucleotide have been solved at high resolution using heteronuclear NMR techniques These approaches require the preparation of uniformly labeled N- and C-RNA oligonucleotides via in vitro transcription with labeled nucleoside triphosphates. The use of RNAs labeled with NMR-active nuclei allows for the application of sophisticated heteronuclear NMR methods. These methods facilitate the assigmnent of NMR resonances and the acquisition of structural restraints for detailed structure determinations. 

E. Trevisiol, E. Defrancq, J. Lhomme, A. Laayoun, P. Cros, Synthesis of nucleoside triphosphates that contain an aminooxy for post-amplification labelling, Eur. J. Org. Chem. (2000) 211-217. 

Before the sequencing begins it is necessary to prepare a short primer that is complementary to a sequence at one end of the DNA strand to be sequenced. This may be prepared enzymatically,622 623 or by non-enzymatic synthesis. The short primer is annealed to the end of the DNA and the resulting molecule is incubated with a DNA polymerase and a mixture of the four mononucleotide triphosphates, one of which is radio-labeled in this position. Four reaction mixtures are prepared. Each mixture contains all four nucleoside triphosphates and also one of four different chain-terminating inhibitors, the most popular of which are the 2, 3 -dideoxyribonucleoside triphosphates ... 

The use of autoradiographic methods confirmed bidirectional replication. Strains of amino acid aux-otrophs with small nucleoside triphosphate pools were used. The addition of amino acids after starvation led to initiation of replication with only a 6-min lag. The cells were labeled with [3H]thymidine, and after the replication forks had moved a short distance from the origin of replication the cells were given a pulse of "super-hot" [3H]thymidine. Using autoradiography it was possible to observe the clearly bidirectional replication forks378 (Fig. 27-17). Replication in other bacteria is also bidirectional. 

A typical experiment is sketched out in Fig. 3. A 32P-labeled nucleotide is used to measure the amount of newly synthesized RNA as a function of time. The concentrations of the substrate (the four nucleoside triphosphates of A, U, G, and C) and that of the enzyme are constant. The initial template concentration is varied by serial dilution with a constant dilution factor. The increase in RNA concentration in the course of time can be divided into three phases ... 

Enzymatic labeling using any of these polymerase methods results in derivatized nucleoside triphosphates being incorporated at numerous locations within an oli-... 

Under these conditions the octanucleotide linker fragments chromatograph at a similar rate to nucleoside triphosphates. There is usually sufficient of the labelled triphosphate, used for the 3 -end labelling, remaining in the sample to act as a marker for the linker peak which elutes later. The first fractions are enriched in the longer DNA fragments—which tend to clone with lower efficiency—and this crude fractionation decreases the number of random plaques which have to be picked to obtain cloned representatives of all the fragments. 

Restriction enzymes which cleave double-stranded DNA, making staggered cuts which leave the 5 -end of each strand extended. Double-stranded DNA with protruding 5 -ends is more efficiently phosphorylated by polynucleotide kinase than DNA with flush or recessed 5 -ends. The fore-shortened 3 -ends can also be labelled by extending them with 32P-labelled nucleoside triphosphates complementary to bases in the extended template strand using DNA polymerase. Y—pyrimidine nucleotide, R—purine nucleotide, N—any nucleotide. A complete list of commercially obtainable restriction endonucleases is given in Appendix III. 

Burgess reported the synthesis of a set of energy transfer dye-labeled nucleoside triphosphates (254a-f). To achieve this synthesis, the coupling of the dye to the nucleosidic moiety had to be performed after formation of the triphosphate esters. 

---