Nick-translation method

DNA polymerase 1. For most applications DNA polymerase 1 from E. coli is used. The enzyme attaches to a short single-stranded region in a dsDNA molecule and then synthesizes a new strand of DNA, degrading the existing strand as it proceeds. When used in vitro this incubation is carried out at 12-15 °C to prevent more than one round of replication occurring. It is used for in vitro labelling of DNA by the nick translation method (described below). 

The nick translation method can be used in conjunction with cell-cycle analysis to determine which phases of the cell-cycle the apoptotic cells are in. To do this, add 5 pg/mL propidium iodide to the samples 30 min prior to analysis. 

As mentioned at the outset, this simplified procedure may not be as consistently reliable as the slightly longer Maat and Smith method. It is capable of giving excellent results, however, and if a nick translation method is chosen as the sequencing tool it may be worth attempting a one-off experiment using the simplified procedure described. 

This recently described procedure is essentially a marrying of the Maat and Smith (1978) nick translation method and the plus and minus method of Sanger and Coulson (1975). The reason for this development was to circumvent one recurrent problem of the nick translation methods, namely the uneven distribution of band intensities obtained particularly when a row of repeated nucleotides... 

The DNA probes are often labeled by the nick-translation method.21 This involves the combined activities of three enzymes DNasel, 5, 3 -exonuclease and... 

Figure 9.17. Labeling DNA probes by the nick-translation method.

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. 

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

The modified DNA may be recovered by alcohol precipitation according to the method in Section 1 (this chapter) described previously for nick-translation modification. Alternatively, dialysis or gel filtration may be done to remove excess reactants. 

In addition to biotin, a digoxigenylated derivative of dUTP was also synthesized. This derivative of dUTP can be incorporated into DNA by Pol I (or the Klenow fragment of Pol I). Therefore, digoxigenin-labeled DNA probes can be prepared by nick translation or random primed-labeling methods developed for the biotin system. It is almost certain that more nonradioactive alternatives to biotin and digoxigenin will be developed in the future. Chemiluminescent methods for nonradioactive probe detection are now widely being used... 

Nick translation is probably the most sensitive method to prepare nonradioactive DNA probes In general, it is simple, easy, and reasonably reliable. However, m order to achieve reproducible results, use of reagents and DNA samples of the highest quality is strongly recommended... 

This method can only be used to label linear DNA molecules It is particularly useful for the DNA samples extracted from agarose gels, especially short DNA fragments, for which nick translation usually gives poor results... 

Detailed protocol for nick-translation sequencing method... 

Preparation of Probe. Probes for genomic Southern blots have been made by a variety of methods and must be of high specific activity. A simple method that yields a probe with a very high specific activity utilizes the polymerase chain reaction (PCR).16 An advantage of PCR over methods based on nick translation or primer extension is that only one strand of DNA is synthesized, so that reassociation of denatured single-strand probe in solution is minimized. 

The end product of the random-primed labeling reaction is a mixed population of unlabeled and labeled strands. Even if the reaction goes to completion, the labeled (synthesized) strands only account for 50% of the DNA strands. This is probably the reason why probes labeled by this method are not as sensitive as optimally nick-translated probes. However, this method is relatively more reliable and reproducible than nick translation. This method can only be used to label linear DNA molecules. It is particularly useful for the DNA samples extracted from agarose gels, especially short DNA fragments, for which nick translation usually gives poor results. 

Biotin-1 l-deoxyuridine-5 -triphosphate (BiodUTP) and reagents for its incorporation into DNA by nick translation are obtained commercially. The products from Bethesda Research Laboratories (BRL, Gaithersbui, MD) are acceptable. BRL now provides a prepackaged kit (BioNick) containing necessary supplies and employing biotin-14-dATP as the source of biotin. The concentration of the resulting biotinylated DNA is determined by the histochemical method for biotin (below). Adequate instructions are provided with these kits. 

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