Many new nucleic acid-based diagnostic tools or assays have been developed that allow analysis of DNA and RNA molecules in clinical samples. These assays are now routinely used for monitoring or detecting, as well as to help decide which therapies would work best for patients. Specific molecular probes and primers are designed for this purpose. Gene probes are used in various blotting and in situ hybridization (ISH) techniques for the detection of nucleic acid sequences in food industry, environmental, medical, and veterinary applications to improve the specificity of the analyses. In medicine, they can help in the identification of microorganisms and the diagnosis of infectious, inherited, and other diseases. In practice, double and single stranded DNAs, mRNAs, and other RNAs synthesized in vitro are all used as probes. DNA/RNA probe assays are faster and sensitive so that many conventional diagnostic tests for viruses and bacteria involving culturing of the organisms are being fast replaced by molecular probe assays. While culture tests can take days, molecular probe assays can be performed within a few hours or minutes. Molecular probes can be broadly categorized into DNA probes and RNA probes, cDNA probes, and synthetic oligonucleotide probes.

Nucleic acid probes are either a single stranded DNA or RNA with a strong affinity towards a specific DNA or RNA target sequence. This affinity and complementary sequence allow binding to specific regions of a target sequence of nucleotides. The degree of homology between target and probe results in stable hybridization. In developing a probe, a sequence of nucleotides must be identified, isolated, reproduced in sufficient quantity, and tagged with a label that can be detected. In theory, any nucleic acid can be used as a probe provided it can be labeled to permit detection and quantitation of the hybrid molecules formed between the probe and sequence to be identified.

What are DNA probes?

A DNA probe is a labeled fragment of DNA that contains a nucleotide sequence specific for the gene or chromosomal region of interest. A variety of methodologies for labeling DNA are used to generate end-labeled or continuously labeled probes. Most enzyme-mediated labeling techniques are very much dependent on polymerase activity, which is responsible for incorporation of the labeled nucleotides. Furthermore, the use of Taq or other thermostable DNA polymerases permits labeling reactions to be performed at higher temperatures via PCR, thereby reducing the incidence of enzyme-mediated point mutations during probe synthesis. PCR is an excellent method for probe synthesis, requiring very small quantities of template material. In the presence of appropriately labeled nucleotide primers, PCR products are labeled as they are being synthesized. Alternatively, the primers themselves may be labeled non-isotopically during their own synthesis, negating the requirement for the inclusion of labeled nucleotide precursors as part of the reaction mix. Random priming is a type of primer extension in which a mixture of small oligonucleotide sequences, acting as primers, anneal to a heat-denatured double-stranded template. The annealed primers ultimately become part of the probe itself, because the Klenow fragment of DNA polymerase I extends the primers in the 3′ direction and, in so doing, incorporates the label. Nick translation (NT) is one of the oldest probe labeling techniques. It involves randomly nicking the backbone of a double-stranded DNA with dilute concentrations of DNase I. At extremely low concentrations, this enzyme nicks a template at four or five sites, producing a free 3′-OH group that can act as a primer at each nicking location. Next, the enzyme DNA polymerase I removes the native nucleotides from the probe molecules in the 5′→3′ direction (exonuclease activity) while replacing them with labeled dNTP precursors by virtue of its 5′→3′ polymerase activity. Nick translation is efficient for both linear and covalently closed DNA molecules, and labeling reaction are completed in less than an hour.
Enzo offers a Nick Translation DNA Labeling System 2.0 to provide a simple and efficient method for generating labeled DNA. The kit can accommodate a wide range of fluorophore-labeled, biotin-labeled, and digoxigenin-labeled nucleotides. In addition to the choice of label, the kit design allows the user to optimize incorporation and product size by adjusting the ratio of labeled-dUTP to dTTP. The ready-to-use NT Enzyme Mix is user friendly and minimizes error from pipetting. Probes labeled by nick translation can be used in many different hybridization techniques including: chromogenic in situ hybridization (CISH), fluorescent in situ hybridization (FISH), screening gene banks by colony or plaque hybridization, DNA or RNA transfer hybridization, and re-association kinetic studies.

What are RNA probes?

RNA probes are stretches of single-stranded RNA used to detect the presence of complementary nucleic acid sequences (target sequences) by hybridization. Compared to the diverse methods for DNA probe synthesis, there is only one reliable method for labeling RNA probes, namely in vitro transcription. It is a reliable and economical method for generating RNA probes. Large amounts of efficiently labeled probes of uniform length can be generated by transcription of a DNA sequence ligated next to an RNA promoter. One excellent strategy is to clone the DNA to be transcribed between two promoters in opposite orientations. This allows either strand of the cloned DNA sequence to be transcribed in order to generate sense and antisense RNA for hybridization studies. One alternative method to generating continuously labeled RNA probes by in vitro transcription is to label the 5’ end of the molecule. This method of 5’ end-labeling is colloquially known as the kinasing reaction. This reaction specifically involves the transfer of the γ phosphate of ATP to a 5’-OH substrate of RNA or DNA (forward reaction). The forward kinasing reaction is far more efficient than the exchange reaction that involves the substitution of 5’ phosphates.
Probe synthesis by 3’ end-labeling involves the addition of nucleotides to the 3’ end of DNA. 3’ end-labeling of DNA is most often catalyzed by terminal transferase. Single- and double-stranded DNA molecules are labeled by the addition of dNTP to 3’-OH termini. RNA can also be 3’ end-labeled using poly(A) polymerase. This enzyme, which is naturally responsible for nuclear polyadenylation of many heteronuclear RNAs, catalyzes the incorporation of adenosine monophosphate. In addition to its utility in RNA probe synthesis reactions, poly(A) polymerase can be used to polyadenylate naturally poly(A)-mRNA and other RNAs.


in vitro transcription is a reliable and economical method for generating RNA probes. Large amounts of efficiently labeled probes of uniform length can be generated by transcription of a DNA sequence ligated next to an RNA promoter. One excellent strategy is to clone the DNA to be transcribed between two promoters in opposite orientations. This allows either strand of the cloned DNA sequence to be transcribed in order to generate sense and antisense RNA for hybridization studies. One alternative method to generating continuously labeled RNA probes by in vitro transcription is to label the 5′ end of the molecule. This method of 5′ end-labeling is colloquially known as the kinasing reaction; it specifically involves the transfer of the γ phosphate of ATP to a 5′-OH substrate of RNA or DNA (forward reaction). The forward kinasing reaction is far more efficient than the exchange reaction which involves the substitution of 5′ phosphates.

Probe synthesis by 3′ end-labeling involves the addition of nucleotides to the 3′ end of either DNA. DNA 3′ end-labeling is most often catalyzed by terminal transferase. Single- and double-stranded DNA molecules are labeled by the addition of dNTP to 3′-OH termini. RNA can also be 3′ end-labeled using the enzyme poly(A) polymerase. This enzyme, which is naturally responsible for nuclear polyadenylation of many heteronuclear RNAs, catalyzes the incorporation of Adenosine Mono Phosphate. Isotopic labeling requires α-labeled ATP precursors. In addition to its utility in RNA probe synthesis reactions, poly(A) polymerase can be used to polyadenylate naturally poly(A)– mRNA and other RNAs in order to support oligo(dT) primer-mediated synthesis of cDNA.

What are Enzo’s AMPIVIEW™ RNA probes?

AMPIVIEW™ RNA probes are designed with Enzo’s patented LoopRNA™ technology and hybridize to a specific RNA, DNA, or RNA and DNA target of interest. The probes consist of oligonucleotides designed for specific targets and offer unbiased signal amplification from the loop in the probes. These loops are non-hybridizing biotin- or digoxigenin-labeled regions that enable the associated target to be visualized in a specific color after chromogenic development (Fig. 1).




AMPIVIEW™ RNA probes are uniquely designed with the precision of targeted, sequence-specific RNA probes and enhanced sensitivity for the detection of nucleic acid targets in cells and tissue, while preserving the morphology of the sample. They are compatible with existing biotin- or digoxigenin-based detection assays and do not require specialized instrumentation. Importantly, they do not rely on lengthy protocols typically required with branched DNA probe technology, thereby diminishing potential background. Scientists can, therefore, visualize the expression and spatial localization of their targets with ease.

Use of Probes in Research Applications

In Northern blotting, RNA is fractionated by gel electrophoresis. The molecules are then transferred to a membrane that is incubated with the labeled probe(s). Hybridization of complementary sequences allows visualization of target RNA sequence. Southern blotting involves the fractionation and transfer of DNA to membranes. Membranes are then incubated with the labeled DNA probe(s). Hybridization of complementary sequences allows visualization of target DNA sequence.

CISH and FISH experiments allow for the localization of RNA or DNA targets in cells and tissues. This technique uses cultured cells or tissue section samples for hybridization and detection of the gene or target sequence of interest. Cells or tissues are processed so that their endogenous nucleic acids are fixed in place, but available for hybridization to and detection by labeled probes. Advances in spatial biology and single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. Techniques for profiling and understanding RNA expression at single-cell resolution have rapidly progressed in recent years.

Enzo Life Sciences is a recognized global leader in providing DNA and RNA labeling technologies with several key patents in developing biotin, digoxigenin, and fluorescent labeled nucleotide probes for gene- and gene expression-related studies. We offer a range of products for in situ hybridization (Fig.2).




For a simple and efficient method for generating labeled DNA, please check out our Nick translation DNA labeling kit, Bio-16-dUTP, digoxigenin-dUTP, as well as our SEEBRIGHT® fluorescent dye-dUTPs. For visualizing the expression and spatial localization of target genes, please have a look at our AMPIVIEW™ RNA probes webpage for further information. Enzo can design probes for practically ANY gene in ANY genome to be used in ANY tissue or cells. To order or inquire about custom probes, please fill in the form and a product specialist will contact you. For all questions and concerns regarding any of our products, our Technical Support Team is here to assist.

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