Random-priming method

Label 50 ng probe using the random prime method according to the manufacturer s directions. 

A convenient random-priming method is labeling on a nylon membrane. Template can be spotted on the membrane, but it is also possible to use DNA restriction fragments transferred after electrophoretic separation onto nylon membranes (Chapter 9). DNA fixed on nylon membranes can serve as a template and the unincorporated precursors can be removed by simple washing for 1-2 min. The probe is then eluted from the membrane in formamide or in water. These membrane-bound DNAs can be reused. The probes synthesized by this method are as efficient in detecting nucleic acid as those synthesized in solution (Bhat, 1990). Similar methods have been proposed earlier for the synthesis of ij DNA probes from M13 templates (Ashley and MacDonald, 1984 Hansen et al., 1987). 

The whole coding sequence is labeled by incorporation of [a- P]dATP using the random priming method and the specific kit purchased from Roche Molecular Biochemicals (Mannheim, Germany). The hybridization of the whole coding... 

Analv.si.s of mRNA levels and chromosomal location of genes Isolated polyA+ RNA was electrophoresed under denaturing conditions and transferred to a nylon membrane filter. Restricted genomic DNA was fractionated in agarose gels and also transferred to a nylon membrane (2). Probes were labelled with P using a random priming method. 

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... 

Related introns with considerable sequence similarity may be detected by heterologous hybridization with labeled probes. Restriction fragments of suitable length to cover almost the entire sequence of a cloned intron are labeled radioactively or by alternative, nonradioactive methods. We use the following protocol for random primed DNA labeling, derived from Feinberg and Vogelstein.14... 

Biotinylated dUTP can also be used to label DNA probes by a different method, namely random-primed labeling (4). The principle of this method is based on the reannealing of hexadeoxyribonucleotide primers, which have random specificity, to the denatured DNA strands. The DNA to be labeled has to be linearized and denatured before the strands are used as templates in the labeling reaction. The complementary strands are synthesized from the 3 OH termini of the reannealed hexanucleotides by the Klenow fragment of E. coli DNA polymerase I. The primers reanneal at random sites of the template strands, so that the synthesis of the complementary strands is primed at random sites. If one of the deoxyribonucleoside triphosphates present in the reaction mixture is labeled, the newly synthesized strands will become labeled by the incorporation of the labeled nucleotides. The end product of this reaction is a mixture of unlabeled (template) and labeled... 

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. 

The convenient solid phase method of Bhat (1990) starts with the denaturation of template (plasmid) DNA in 0.5 N KOH at room temperature for 5 min. Samples of 1-2 xl (100-200 ng) are then spotted on 3 X 3 mm pieces of nylon membrane. The DNA membrane can be neutralized by soaking in 2 M Tris-HCl (pH 7.4) or used directly for DNA binding, e.g., by UV-binding for 3 min on a transilluminator or by incubation at 80°C for 1-2 h. A random priming mix (50 jxl) with all the reagents (except enzyme) is placed into an Eppendorf tube and one nylon membrane square with DNA is added. After 5 min, enzyme is added and the reaction left to proceed for 4 h. The filter bit is then removed and left immersed in 2 X SSC (Table 8.4) for 10 s (5 times, each in a fresh 1(X) ml). The final wash was in 1 ml of H2O in an Eppendorf tube and heated for 5 min at 9O C (no more than 2% of probe remains bound and the filter is removed for reuse at this point). About 60% of the precursor is chased into the probe. 

The upstream primer (5 -CACAATTCCACACAAC) binds upstream of the insert and is extended by DNA polymerase (Klenow) away from the insert. Any labeled dNTP, as in random priming, can be used to obtain a probe with reasonably high activity. Under the experimental conditions (Brown et al., 1982 Hu and Messing, 1982), the synthesis is not allowed to reach completion to leave the insert region S5. After phenol extraction and spin chromatography on Sephadex G-50, the probe is ready for use (no heat-denaturation ). This method is extremely simple and yields strand-specific probes. 

For nonoligonucleotide probes, in vitro transcription (riboprobes), random-primed labeling, nick translation, and PCR-amplification (all for DNA probes) could be the methods of choice for probe labeling with either radioactive or non-radioactive labels. Unless target sequences are not abundant (neuropeptide mRNAs usually are abundant), the use of nonradioactively labeled probes is highly recommended because of a Speed and ease of detection b. Supenor resolution ... 

Run a 1% agarose gel to assess cDNA quality (see section 3.3.4). Label the cDNA by the random-primed Klenow method (section 3.3.5). 

This is a further method for producing cDNA (double-stranded in this case) suitable for labeling by the random-primed Klenow method. 

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