Hybridization nick translation

Constraints imposed by the intended application may limit the choice of labeling methods (Table 7.2). For example, when the probe should be intact, such as in Sj mapping, labeling by nick translation should be avoided. In contrast, for most slot blot and in situ hybridizations, nick translation or random primer extension methods provide suitable probes with an optimum label density, often with the added benefit of ss extensions on the duplex offering the possibility of amplification through hyperpolymer formation. 

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. 

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. 

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-... 

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. 

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. 

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... 

In addition to the detection of antigens and antibodies, EIA will, undoubtedly, play an increasingly important role in molecular biology. For example, the bio-blot method (Leary et al., 1983) for the detection of DNA-DNA or DNA-RNA duplexes on nitrocellulose membranes offers important advantages over conventional procedures in which radioactive probes are used and autoradiographic detection. In this method, biotinylated DNA probes are prepared by nick translation (Rigby et al., 1977) in the presence of biotinylated analogs and hybridized with the DNA or RNA on filters. Biotin is then detected by avidin-labeled enzyme (Section 3.1). 

Sometimes, e.g., to calibrate nick translation reagents or to analyze the size of cDNA in SI resistant DNA RNA hybrids, it is... 

Labelling. For each hybridization mixture digest l tg of purified reference DNA with HaelU restriction enzyme and label this with dCTPp ] by nick translation according to the manufacturer s instructions. 

Total RNA was isolated as Wallace (19), and poly(A )-RNA purified using oligo-dT-cellulose. Northern blot analysis were performed by e-lectrophoresis of the RNA in formaldehyde-MOPS agarose and blotted onto Gene Screen membranes. trxA gene labelled with P-dCTP by nick-translation was used as a probe. Methods used for hybridization and washing of filters were those recommended by the manufacturers. 

Fig. 5. Analysis of RNA complementary to MMTV DNA in 341 cells treated with (ADP-ribose) synthetase inhibitors. Total RNA from cells treated with 10 tM 3-ABm for 0 lanes a, f), 4 (lane b), 16 (lanes c, h), 32 (lane d), and 64 h (lane e), or with 10 mM 3-aminobenzoic acid for 16 h (lane g), was subjected to electrophoresis on 1.2% agarose gels, transferred to a nitrocellulose paper, and hybridized with nick-translated P]-labeled MMTV DNA specific for the env region. The size of the RNA were extrapolated from the migration of radiolabeled Hind III DNA standards (lane i) (4.4, 2.3, and 2.0) kilobase pair (Kbp) and 18S and 28S rRNA from the ethidium bromide staining pattern...

Analysis of the cloned DNA by dot hybridization. The single stranded DNA of each Ml 3 clone was spotted onto a nylon membrane (Hyband-N, Amersham). Hybridizations were done according to the supplier of the nylon membrane using nick translated P-labeled probes. The probes were either the "140 bp DNA" (Fig. 2A), the "70 bp DNA" (Fig. 2B) or the total... 

Gel blot hybridizations 20 pg of purified mutant or wild-type DNA were digested with one or more restriction enzymes, electrophoresed through 0.8% or 1.5% agarose gels, and transferred to nitrocellulose or DBM paper according to the method of Southern or Wald et al. Hybridization was carried out initially under aqueous conditions and later with the dextran sulfate modification, using nick-translated aprt DNA as a probe. 

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. 

---