Random primed labeling

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... 

The end product of the random-primed labeling reaction is a mixed population of unlabeled and labeled strands Even if the reaction achieves completion, the labeled (synthesized) strands only account for 50% of the DNA strands This is probably... 

To enable a covalent labeling of target molecules, the dyes are functionalized with reactive groups. These are either phosphoramidites for the labeling of synthetic oligonucleotides (solid-phase synthesis), or uridine-5 -triphosphates for enzymatic coupling in PCR (random primed labeling... 

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. 

K9. Kincaid, R. L., and Nightingale, M. S., A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling. BioTechniques 6, 42-49 (1988). 

Large quantities of hybridization probes can be prepared from small amoimts of DNA by filter priming the DNA fragment is immobilized on a nylon filter using UV light and used as template to prepare radioactive probes in a random primed labelling reaction.309 probes are removed from the filter by heating, purified and used, while the filter-bound DNA may be re-used. 

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 ... 

The probe DNA is labeled by incorporation of the thymidine analog digoxigenin-11-dUTP by the random primed labeling technique of Feinberg Vogelstein (1983). 

Dry down 50 il of tritiated thymidine (Amersham TRK. 576 in EtOH water 1 1) in each of 2 Eppendorf tubes. Each 50pl will be used for 1 reaction. We use the Boehringer random primed labeling kit (Cat. 1004760) with some modification to increase the average size of the product. To each vial with the dried hot TTP add ... 

Random Prime Labeling of DNA Probes with Fluorescein-11-dUTP... 

Reagents 1-5 are available, fully optimized, from Amersham International pic as ECL random prime labeling and detection system (RPN 3030/3031/3040). The system is extensively tested and necessary controls are also provided. The reagents are stable for at least 3 mo. 

Fig. 4. Kinetics of random primed labeling with fluorescein-11-dUTP The yields given are total amount of DNA present (labeled DNA plus template) following each standard labeling reaction. Incubation at room temperature.

---