Labeling by random priming

Fig. 8.2. The detection of membrane-bound sequences (shown for a tailed probe in 1) can be modified to considerably enhance the signal. One system (II) consists of target-specific primary probes and labeled, target-independent secondary probes. The secondary probes are specific for vector sequences of the primary probe. It is possible to have multiple primary probes, each recognizing a different region of the target. If the primary probes are M13, then all M13-specific secondary probes serve as universal probes. This patented system is marketed by ImClone Systems as AmpIiProbe. Primary probes, labeled by random priming or nick-translation, also yield hyperpolymers (III) and hence a signal amplification. The current development of branched DNA could lead to a replacement of the hyperpolymer systems. Probes with partial overlaps, e.g., obtained with nick-translation, may also form hyperpolymers.

This amplified cDNA is then run on a 1% agarose gel for quality control and labeled by random primed Cy-dye incorporation using Klenow fragment (see section 3.3.5). 

Prepare a 32P-labeled probe by random priming (Amersham Megaprime DNA Labeling System) using a portion of the HBV genome as a template (see Note 4). Purify the probe through a Clontech Chroma Spin column as directed by the manufacturer. 

Fig. 7.14. Preparation of labeled probes by random priming. Both ss and ds DNA can serve as a template.

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). 

There are a number of high-quality kits available for labeling DNA by random priming. If you choose a commercial kit, be sure that it can be adapted to use two radionucleotides, since the probe must be of very high-specific activity to detect rare clones. 

Reverse Transcription (to Be Followed by Random-Primed Klenow Labeling)... 

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... 

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. 

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. 

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 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. 

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. 

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... 

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