What is the difference between probes and primers

Progress in sequence-specific DNA imaging by fluorescence microscopy has been achieved by employing the fluorescent hybridization in situ FISH method.

This type of label is especially useful for the direct examination of microbiological or cytological specimens under the microscope.

Figure 1: Fluorescence emission profiles of available fluorescent labeled dUTPs. The dye-dUTPs are designed to perform especially well in multi-color applications, such as in situ hybridization and microarray analysis. What are DNA probes? A DNA probe is a fragment of DNA that contains a nucleotide sequence specific for the gene or chromosomal region of interest. DNA probes employ nucleic acid hybridization with specifically labeled sequences to rapidly detect complementary sequences in the test sample.

A variety of methodologies for labeling DNA have been described. In short, these methods 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. Nick translation is one of the oldest probe labeling techniques. Nick translation is efficient for both linear and covalently closed DNA molecules, and labeling reaction are completed in less than an hour. The kit can accommodate a wide range of fluorophore-labeled, biotin-labeled, and digoxigenin-labeled nucleotides.

In addition to 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: in situ hybridization ISH , 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. RNA probes are usually labeled, for example with radioisotopes, epitopes, biotin or fluorophores to enable their detection.

RNA probes as hybridization tools remain popular because of several key advantages associated with their use. These probes are synthesized by in vitro transcription and can be substituted for DNA probes in nearly all applications.

RNA probes are single-stranded and offer several advantages over DNA probes including improved signal or hybridization blots. Compared to the diverse methods for DNA probe synthesis, there is only one reliable method for labeling RNA probes, namely in vitro transcription.

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.

This enzyme, which is naturally responsible for nuclear polyadenylation of many heteronuclear RNAs, catalyzes the incorporation of Adenosine Mono Phosphate.

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. 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. Figure 2: Enzo Life Sciences offers a complete set of solutions for in situ hybridization, providing everything you need for labeling, hybridization, and detection. Advances in 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 and fluorescent labeled nucleotide probe for gene expression studies. We offer a range of products for Genomics research needs. In qPCR, probes are labeled with fluorescent dyes or radioactive elements. These probes are hybridized with the target sequence in the DNA duplex.

Different types of labeled probes, either with radioactive elements or fluorescence, are used in various types of hybridization reactions as well. The hybridization of the PNA probes to their target sequences are shown in figure 1. PNA probes are used to determine the length of the telomeres.

During hybridization, probes bind to the single-stranded DNA in a complementary manner. The target sequence is flanked by two primers known as forward primer and reverse primer. Specificity and complementarity are the primary factors in the designing of primers.

Secondary structures should also be avoided. Other factors that should be considered during the designing of primers are described below. The annealing of the forward and reverse primer to the two strands of the target DNA is shown in figure 2. This section describes the various types of fluorescent dyes, PCR primers, and probes, and their specific uses in real-time PCR experiments. Therefore, the overall fluorescence signal from a reaction is proportional to the amount of dsDNA present and increases as the target is amplified and more PCR product accumulates.

As target amplification proceeds exponentially until the DNA primers are depleted, the fluorescence signal also increases exponentially, forming the basis for calculation of the original template quantity by the extrapolation of the real-time amplification curves. DNA-binding dyes can be classified as either nonsaturating or saturating. Nonsaturating dyes — for example, EvaGreen — do not inhibit PCR and can be used at a sufficiently high concentration to ensure that all vacant binding sites on dsDNA are bound by dye.

However, saturating dyes are preferred for use in high resolution melt assays due to the more discrete signal change observed upon DNA denaturation see High Resolution Melting HRM. The advantages of using dsDNA-binding dyes include simple PCR primer design only two sequence-specific DNA primers are needed, so probe design is not necessary , the ability to test multiple genes quickly without the need for multiple probes for example, in the validation of gene expression data from many genes in a microarray experiment , lower initial cost than probes, and the ability to perform a melt curve analysis to check the specificity of the amplification reaction.

As a result, the presence of nonspecific products in a real-time PCR reaction, such as PCR primer-dimers, contributes to the overall fluorescence and reduces the accuracy of quantification. Furthermore, DNA-binding dyes cannot be used for multiplex reactions because fluorescence signals from different products cannot be distinguished when, for example, amplifying a control gene along with a target gene using different PCR primers in the same reaction mixture.

Many fluorescent PCR primer- and probe-based chemistries have been devised and are available from different commercial vendors, including:. PCR primer- and probe-based detection chemistries share some common features. In general, these chemistries use some form of fluorescence quenching to ensure that target-specific fluorescence is detected only when amplicon from the product of interest is present. The PCR primer or target-specific oligonucleotide probe is labeled with a reporter fluorophore, and in most cases, its fluorescence is quenched when the specific target DNA sequence is not present.

Usually, this quenching is accomplished by covalently attaching a quencher molecule to the DNA primer or probe in combination with some mechanism by which the reporter and quencher are separated when the primer or probe binds to its specific target sequence.

First, these molecules specifically detect the target sequence, so nonspecific products are not detected and do not affect the accuracy of quantification. Second, the use of fluorescent DNA primers and probes enables single-tube multiplexing of qPCR reactions for multiple target sequences.

Hydrolysis TaqMan Probes Hydrolysis assays TaqMan or 5' nuclease assays include a sequence-specific, fluorescently labeled oligonucleotide probe in addition to a sequence-specific PCR primer. The hydrolysis probe is labeled with a fluorescent reporter at the 5' end and a quencher at the 3' end.

Commonly used fluorescent reporter-quencher pairs are fluorescein FAM , which emits green fluorescence, and Black Hole Quencher 1 dye. Hydrolysis probes are hydrolyzed by Taq polymerase. When the hydrolysis probe is intact, the fluorescence of the reporter is quenched due to its proximity to the quencher Figure 2. As a result, the reporter is separated from the quencher, resulting in a fluorescence signal that is proportional to the amount of amplified product in the sample.

The main advantages of using hydrolysis probes are high specificity, a high signal-to-noise ratio, and the ability to perform multiplex reactions. The disadvantages are that the initial cost of the probe may be high, and the assay design may not be trivial.

Molecular Beacons Molecular beacon assays include a sequence-specific, fluorescently labeled oligonucleotide probe called a molecular beacon. A molecular beacon is a dye-labeled oligonucleotide 25—40 nt that forms a hairpin structure with a stem and a loop Figure 3.

The 5' and 3' ends of the probe have complementary sequences of 5—6 nucleotides that form the stem structure. The loop portion of the hairpin is designed to specifically hybridize to a 15—30 nucleotide section of the target sequence.

A fluorescent reporter molecule is attached to the 5' end of the molecular beacon, and a quencher is attached to the 3' end. Formation of the hairpin therefore brings the reporter and quencher together, so no fluorescence is emitted. The stem-loop structure of molecular beacons brings the fluorophore and quencher together. During the annealing step of the amplification reaction, the loop portion of the molecular beacon binds to its target sequence, causing the stem to denature. The reporter and quencher are thus separated, quenching is abolished, and the reporter fluorescence is detectable.

Because fluorescence is emitted from the probe only when it is bound to the target, the amount of fluorescence detected is proportional to the amount of target in the reaction. Molecular beacons have some advantages over other probe chemistries. They are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur, which is especially desirable for allelic discrimination experiments.

Unlike hydrolysis assays, molecular beacons are displaced but not destroyed during amplification because a DNA polymerase lacking 5' exonuclease activity is used. The main disadvantage of using molecular beacons is that they are difficult to design.