Probe nick translation

Nick translation is one of the DNA-labeling techniques conventionally used to prepare hybridization probes. Nick translation utilizes combined activities of DNase I which introduces nicks (or single-strand breaks) and the 5 -(exo)nuclease and polymerase activities of E. coli DNA Pol I. While the 5 -nuclease activity of Pol I removes the nucleotides from the 5 -phosphoryl terminus, the polymerase activity carries out the sequential addition of nucleotides to the 3 -hydroxyl terminus, thus translocating the nick. When a highly radioactive nucleotide, e.g., [o - P]dATP, is included during the reaction, nick translation results in the uniform labeling of duplex molecules with a specific activity >10 cpm//zg DNA (1). Nick translation produces labeled DNA probes from both strands. Pol Ik, which lacks 5 -nuclease activity, cannot perform the nick translation, but it can carry out a strand displacement synthesis. 

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. 

Purify the labeled probe by ethanol precipitation according to steps 4-7 of the protocol previously described for nick translation. 

Isolate the labeled probe by alcohol precipitation as described previously for nick translation. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 for nick translation (this chapter). 

Hapten-labeled DNA probes for FISH assays can be used for BISH assays and those probes can be purchased from various vendors. If commercial hapten-labeled DNA probes are not available, DNA probes can be labeled with haptens by nick translation or by PCR in a laboratory. Nick translation kit can be utilized for labeling DNAprobes with hapten (10976776001, Roche Applied Science, Germany). New probes must be analyzed for the specificity by FISH or BISH assays using a comparative genomic hybridization metaphase control slide. 

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... 

Label 1.0 pg of probe with biotin-11-dUTP by the nick translation reaction... 

Biotin- and/or digoxigenin-labeled probes are stable for more than a year when stored at 4°C It is therefore possible to nick translate up to 5 pg of the probes and store a good batch. When compared with tritium (5 ng), the amount of probe used is much larger (typically 20-100 ng)... 

Gastric tumor tissue is fixed with 4% neutral formaldehyde for 1 day and embedded in paraffin (Kitayama et al., 2000). Paraffin sections (6 xm thick) are deparaffinized with xylene and rehydrated with ethanol. Centromeric a-satellite DNA probes and locus-specific identifier probes (c-myc and p53) are available from Vyis Inc. (Downers Grove, IL). The probes are labeled with orange (Cy 3) or green (PITC) using digoxigenin-ll-dUTP and nick translation. The sections are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for 10 min. This is followed by treatment with 0.2% pepsin in 0.01N HC1 for 10 min at 37°C, and then exposure to 0.1% NP40/2 X SSC for 10 min at the same temperature. 

If the target sequence is present on the filter in a low concentration, either because it is a low copy number sequence or because a particular group of taxa yield a low concentration of chloroplast DNA in a total DNA extract, a prehybridization/hybridization buffer that contains dextran sulfate can greatly enhance the rate of hybridization (up to 100-fold44 when nick-translated probes are used) and permit detection of the target se-... 

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. 

In the original system, biotin is attached to the deoxy analog of y-UTP via a spacer arm. Biodnylated dUTP is incorporated into DNA strands by a conventional labeling reaction, nick translation, which is also widely used to prepare radioactive probes (3). The presence of a spacer arm between biotin and dUTP separates these two molecules far apart, and thus, reduces the steric hindrance caused between them. Therefore, the efficiency of labeling, hybridization, and detection is greatly increased. 

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-labeled probes are stable for more than a year when kept at 4°C. Therefore, large amounts (up to 5 lg) can be nick-translated and stored at 4°C. Unlike tritium-labeled probes (5 ng), biotin-labeled probes need to be used in larger amounts (20-l(X) ng per slide). Single-stranded DNA and RNA probes in appropriate vectors are believed to be more promising, but have yet to be evaluated for gene mapping. 

Labeling of probes for in situ hybridization relies on the incorporation of either a radioisotopic dNTP (e.g., dCTP), or of a nonisotopic molecule, such as biotin-7-dAlT or biotin-11-dUTP, by either nick translation or random priming. The site (s) of hybridization can then be seen using autoradiography with isotopic probes, or immunocytochemically if biotin is incorporated into the probe DNA. It is with the latter form of in situ hybridization methodology that this chapter is concerned. 

Biotinylation of whole plasmid (probe) or genomic DNA is carried out according to the protocol provided with the Gibco-BRL nick translation kit. It is convenient to biotinylate 2 ig of DNA at each reaction in a total reaction vol of 100 lL. 

Hybridization solution 45% Formamide, 5X SSC, 5X Denhardt s solution, 20 mMsodium phosphate, pH 6.5, 300 pg/mL freshly denatured, sheared, herring sperm DNA, 200 ng of biotinylated DNA/mL. Before its addition, the biotinylated probe DNA is denatured by incubating for 10 min in a boiling water bath and quickreduce size is unnecessary, since the products generated by nick translation are sufficiently small. Filter and store the hybridization solution as was done for the prehybridization solution. Hybridization solution can be recovered after use and stored at-20°C. The solution can be reused at least 10 times over a time-span of at least 5 mo, without noticeable... 

Nick translation. Suppose that you wish to make a sample of DNA duplex highly radioactive to use as a DNA probe. 

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