Southern and Northern Blot Hybridization

FastTag®-, EndTag™ -, and PHOTOPROBE® Biotinlabeled probes can be used to detect targets on Southern and northern blots as well as colony or plaque lifts. The same well established protocols for radioactive hybridization are employed but without the hazards and disposal issues associated with radioactivity.

Hybridization of the nucleic acid probe to the target on the blot is simplified using the readyto-use HYBEX™ Hybridization Solution. HYBEX™ Hybridization Solution contains no formamide and can be used with both nylon and nitrocellulose membranes.

DNA Molecular Weight Markers can be used as easy size references. These ladders range from 0.2 to 10 kbps, are available biotinylated or unlabeled, are ready-to-use, and contain loading dye for monitoring electrophoretic mobility.

Chemiluminescence can be documented

The UltraSNAP™ Detection System for Nucleic Acid Blots allows for the convenient detection of biotinylated nucleic acids on nylon or nitrocellulose blots. 32, 40, 54 (Please see protocol on page 20). This kit features the DuoLuX™ Chemiluminescent/Fluorescent Substrate (see page 13), alkaline phosphatase streptavidin, and a specially optimized PolyBlock™ Blocking Reagent and washing solutions developed for high sensitivity and low background. One kit contains sufficient reagents to develop approximately twenty 100 cm2 blots.

Detection is accomplished using biotinylated probes that are recognized by streptavidin coupled to alkaline phosphatase. The probe is visualized by the conversion of the DuoLuX™ Substrate to a luminescent and fluorescent product by a dephosphorylation reaction catalyzed by alkaline phosphatase. The sensitivity is enhanced with the use of the PolyBlock™ Blocking Reagent and wash buffers included in the kit.

Chemiluminescence can be documented and recorded by exposure to film or by image capture with digital imaging systems. Blots can be exposed to film several times over an extended period of time, with typical film exposure times of 30 seconds to 10 minutes. Sensitivities as low as 100 fg of target nucleic acid can be achieved. Optimal exposure times using digital imaging systems depend on the sensitivity of the instrument. Chemiluminescence can be developed on either nylon or nitrocellulose membranes, although nylon is preferred because of faster signal production.

Alternatively, the fluorescence of the developed substrate can be recorded with a digital imaging system or conventional camera even months after the chemiluminescence has faded. Acquiring an image from the fluorescent signal requires a much shorter exposure time than from the chemiluminescent signal, often just a fraction of a second.

Selected Reagents:

HYBEX™ Hybridization Solution MB-1230 • 200ml
DNA Molecular Weight Markers
Unlabeled MB-1301 • 25µg
Biotinylated MB-1302 • 25µg
10x Casein Solution SP-5020 • 250ml
Animal-Free Blocker™ (5x) SP-5030 • 250ml
UltraSNAP™ Detection Kit MB-6500 • 1 Kit
UltraSNAP™ Accessory Kit (buffers and blocking agent only) MB-6501 • 1 Kit

In Situ Hybridization

The availability of many different labels and the wide range of detection systems allows the flexibility and sensitivity necessary for in situ hybridization (ISH). For example, for fluorescence in situ hybridization, biotin-labeled probes can be detected with fluorescently labeled avidin or streptavidin. For brightfield microscopy, alkaline phosphataseconjugated streptavidin followed by the enzyme substrate, BCIP/NBT, provides a sensitive detection method. Additional color choices are available with the use of the peroxidase-based VECTASTAIN® ABC Kit followed by the appropriate substrate (e.g. TMB, DAB, ImmPACT™ DAB, Vector® VIP, or Vector® NovaRED™).

Haptens such as dinitrophenyl (DNP), fluorescein, and Texas Red®, for example, provide nearly equally sensitive alternatives to biotin. Several detection options are also available for these labels. Antibodies to these haptens, together with many fluorescent and enzymatic detection methods, provide a great array of possibilities for detecting probes.

With the options available, the researcher has substantial flexibility in optimizing experimental systems, especially in cases where endogenous biotin or enzyme activity, or antibody cross-reactivity present potential problems. The available options also allow the simultaneous detection of multiple, differentially labeled probes in a single experiment.

Chemiluminescence can be documented

CGH sum karyogram was created after analyzing PHOTOPROBE® labeled tumor DNA from a small cell lung cancer. Biotin was detected with Vector® Fluorescein Avidin D (green). Chromosomes from 15 metaphase spreads were analyzed for each hybridization. Figure provided courtesy of Dirk Korinth, Konrad Donhuijsen, Ulrike Bockmühl, and Iver Petersen, Institute of Pathology, University Hospital Charité, Berlin, Germany

Optimization of Probe Length for In Situ Hybridization

NICKIT™ p.s.o. has been developed to reduce the size of large DNA probes using a photo/enzymatic procedure without the problem of over- or underdigestion experienced with other methods. A smaller probe size generally results in better signal and lower background for ISH applications, probably due to better tissue penetration of the probe.

NICKIT™ p.s.o. Probe Size Optimization Kit MB-1905 • 1 Kit

Comparative Genomic Hybridization

Comparative genomic hybridization (CGH), based on fluorescence in situ hybridization, is a technique for screening chromosomal imbalances in tumor tissue. Labeled DNA samples from two sources are competitively hybridized onto normal chromosome metaphase spreads: one labeled DNA sample is isolated from tumor tissue, the other, differently labeled DNA from normal tissue. Based on the differential hybridization of the two different sources of DNA, the over- or under-expression of chromosomal regions can be mapped on the normal chromosomes.

PHOTOPROBE® Biotin has been shown to be an effective labeling technique of highly degraded DNA from archival material for this analysis. 26, 27, 59 Often, DNA isolated from formalin-fixed, paraffinembedded archival tissue is reduced in quality, tending to be smaller than 1000 bp. Using the classical method of nick translation, the resulting labeled DNA fragments are usually too small to give a detectable signal. Because labeling with PHOTOPROBE® Biotin is non-destructive, the original, larger fragments of DNA are labeled allowing for the analysis of gains and losses of DNA from the tumor tissue sample. Over-represented chromosomal regions may correspond to tumor promoting genes, and chromosomal deletions potentially contain tumor suppressor genes.

Cellular Localization

FastTag®-, EndTag™ -, and PHOTOPROBE® Biotinlabeled nucleic acids have been successfully employed in the study of intracellular trafficking of gene delivery complexes and nuclear import mechanisms of viral nucleoprotein molecules. Because these labeling systems do not destroy the nucleic acid nor create nascent nucleic acid, the original DNA or RNA can be tagged and traced throughout the experiment.

For example, in the study of gene delivery, plasmid DNA can be tagged with a FastTag® label. Intracellular trafficking of gold-labeled plasmid DNA can be followed under electron microscopy.13, 14 In addition, fluorescent staining patterns of fluorescently labeled plasmid DNA can be followed under confocal microscopy. 12, 42, 61 In this manner, cellular interactions of the plasmid DNA with different gene delivery vehicles and with cytoskeletal components can be studied. Such investigations can yield insights into gene delivery mechanisms. siRNA samples labeled with FastTag® can be used as internal controls to evaluate transfection efficiency or cell viability. 49

In a similar manner, 5’ EndTag™ labeling of nucleic acid is also used successfully in the study of cellular trafficking and localization of nucleic acids and complexes including viral RNA, 35, 36, 44 ribonucleoprotein particles, 60 and oligonucleotide/peptide complexes. 57

Chemiluminescence can be documented

Cellular uptake of labeled plasmid: FastTag® Fluorescein labeled plasmid DNA (green) incubated with COS-7 cells. Nuclei were counterstained and mounted with VECTASHIELD® with DAPI (blue).

EndTag™-Labeled Primers Used in PCR Amplification

Several applications involve the incorporation of labeled primers into a PCR amplification product:

• Immobilization of DNA target to a solid support for isolating sequence-specific DNA-binding molecules. Synthesizing a DNA fragment by PCR using a labeled primer ensures that the label is located only near the end of the DNA. The target DNA is thus bound to the matrix only at one end, minimizing interference in the binding domains.

• Cloning or library screening by hybrid capture. Immobilized single-stranded DNA can be prepared by PCR amplification in which one of a pair of primers is EndTag™-Biotin labeled. Only a single strand of the PCR product is biotinylated. After binding the labeled DNA to a matrix such as VECTREX® Avidin D, the unlabeled strand can be removed, yielding a matrix ready for hybrid capture.

Covalent Attachment of 5’ EndTag™-Labeled Nucleic Acids to Gold

Nucleic acids containing sulfur atoms can form covalent bonds with gold. The phosphoro-thioate group of 5’ EndTag™-labeled RNA has been employed for immobilization to a gold substrate for atomic force microscopy. 33

Affinity Binding

Genomic/cDNA subtraction, 1, 31, 51 microsatellite isolation, 2, 21, 50, 62 and library screening by hybrid capture24 require the removal or immobilization of nucleic acid hybrids. Affinity binding matrices are available for use with PHOTOPROBE® -, FastTag®-, or EndTag™- labeled probes for such applications.

Irreversible binding of biotinylated nucleic acids to a solid support can be accomplished using VECTREX® Avidin D. Following incubation of VECTREX® Avidin D/biotinylated probe with a cellular extract, hybridized nucleic acids can be recovered by melting the doubled stranded nucleic acid and eluting the unbiotinylated single strands. 4, 23, 41 (Guidelines for this procedure are described on page 26).

Reversible binding of nucleic acids can be achieved with either of two systems:

  1. VECTREX® Avidin DLA matrix has a significantly reduced binding affinity for biotin. Biotinylated nucleic acids that are bound to this matrix can be eluted with biotin. 8
  2. Nucleic acids that have been labeled with FastTag® Fucose or EndTag™ Fucose bind specifically to VECTREX® AAL, a matrix containing the fucosespecific lectin from Aleuria aurantia. These nucleic acids are then easily eluted under mild conditions using the sugar L-fucose at physiological pH and salt concentrations.

Irreversible Binding Matrix
VECTREX® Avidin D A-2020 • 1 ml

Reversible Binding Matrices
For Biotin Labeled Nucleic Acids
VECTREX® Avidin DLA MB-2021 • 1 ml
Binding and Elution Kit MB-2022 • 1 Kit

For Fucose Labeled Nucleic Acids
VECTREX® AAL MB-1396 • 1 ml
VECTREX® AAL Binding and Elution Kit
MB-1397 • 1 Kit

Resolve Same-Sized DNA by Sequence Variation

The ability to resolve DNAs of similar size in a gel is critical for applications such as differential display and Rapid Amplification of Polymorphic DNAs (RAPD) in which multiple DNA species need to be separated before subsequent excision and analysis. Two sequence specific DNA ligands in the Resolve-It™ Kit bind to GC- or AT- rich sites on DNA and retard the electrophoretic migration of DNA in a sequence specific manner. AT-Yellow™, a minor groove binding bisbenzimide-PEG conjugate, binds to AT-rich regions; GC-Red™, an intercalating phenyl neutral red-PEG conjugate, binds GC-regions. A ligand is added to the agarose during gel preparation, and, during electrophoresis, the increased friction caused by ligand association with the DNA retards the mobility of DNA depending on the amount of ligand bound. The resolved bands can be excised and electrophoresed on a gel containing the second ligand, if desired, for confirmation of homogeneity or for additional resolution. The resolved DNA sequences can then be extracted for cloning, sequencing, or other analysis. The quantity of each ligand supplied is sufficient to prepare about 200 ml of agarose gel.

Resolve-It™ Kit - Sequence Specific DNA Ligands MB-1401 • 1 Kit

Reversible binding of FastTag

Reversible binding of FastTag® Fucose-labeled λ Hind III DNA to VECTREX® AAL. Labeled (+) or unlabeled (-) DNA was incubated with VECTREX® AAL (+) or binding buffer (-). Following centrifugation of binding reactions, VECTREX® AAL was washed and the bound DNA eluted with L-fucose. Supernatants and eluates from the binding reactions were fractionated by agarose gel electrophoresis and DNA visualized by ethidium bromide staining. Lane 4 shows that the fucosylated DNA bound to the VECTREX® AAL and lane 8 shows that fucosylated DNA can be easily eluted.

Amplification of Fluorescent Signal in Nucleic Acid Microarrays

Fluorescent amplification of biotinylated probes is common in microarray-based analysis. The multiple binding capability of Biotinylated Anti-Streptavidin provides a method for significant signal amplification. This antibody binds to streptavidin through the antigen binding sites and through the covalently attached biotin residues. Following the first application of a fluorescent streptavidin such as phycoerythrin- or fluorescein-labeled streptavidin, the signal is amplified by incubation with Biotinylated Anti-Streptavidin followed by a second incubation with the fluorescent streptavidin. The same procedure can be performed with labeled avidin and Biotinylated Anti-Avidin. This procedure results in the introduction of more fluorochromes at the target site. 3, 6, 9, 38

Amplification of Signal Using Biotinylated Anti-Streptavidin

Two 600 bp DNA

Two 600 bp DNAs of different sequence form a single band using typical electrophoretic conditions (A). The same DNAs are separated into two distinct bands in a gel containing Resolve-It™ AT-Yellow™ (B)

Amplification of Signal