Random priming

Shao, Z., Zhao, H., Giver, L. and Arnold, F.H. (1998) Random-priming in vitro recombination an effective tool for directed evolution. Nucleic Acids Research, 26, 681-683. 

Although random priming, in general, is less efficient, it may be preferable to oligo dT priming in this instance, because the miR-target mRNA may be significandy deadenylated by miR action (see Fig. 6.3C). 

The first technical ditference between the DASL assay and traditional microarray assays is that a random priming technique is utilized, and as such an intact polyA tail is not required for efficient labeling. Other differences include a mechanism by which two oligonucleotide probes must recognize the RNA of interest in order to be extended, ligated, and later amplified via PCR. This technique leads to both high specificity and high sensitivity (due to the PCR reaction). 

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... 

Of all the possibilities for labeling, the first, DIG random-primed DNA labeling, is usually preferred because of its high yield. 

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... 

An advantage of, 25I is its long half-life (60 days), but the iodination procedure is somewhat difficult to establish. Nick translation and random priming with 32P and 3H are easier to control, but 32P does have a shorter half-life (14 days) than l25I. We prefer nick translation to random priming because it is easier to produce longer tracers. 

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... 

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