Many factors influence the success of DNA hybridization assays, including the length, composition and sequence of the probe, hybridization buffer composition, temperature of hybridization, concentration of target strand and probe, and whether the assay is solution based or solid phase. These factors interrelate and can synergistically effect an assay. For the scientist new to DNA hybridization assays, much is written about the obvious factors which influence the assay, such as probe composition and annealing temperatures, but many other details, some of which are subtle, can greatly influence the sensitivity and quality of data generated from hybridization assays.
DNA hybridization assays once were limited to blotting methodologies as those developed by Southern, but more recently this has been expanded to include reverse capture assays and solution based hybridization assays. The fluorogenic nuclease assay and DNA chips are two examples of technologies which have greatly surpassed the traditional Southern blot.
The following application note is not meant to provide a detailed accounting for performing a hybridization assay, but rather some suggestions on how to improve existing methods and hence the results.
Selecting the Probe
A good probe is single-stranded, specific, and does not self anneal. Though all probes which anneal to their target sequence are single-stranded, they are often generated by random priming or nick translation techniques which will generate homologous probes during the labeling process. The fact that the probe pool contains sense and anti-sense probes can lower the efficiency of hybridization. To avoid probes from re-annealing to themselves, single-stranded synthetic oligonucleotides and RNA probes generated by in vitro transcription can be used.
The sequence and composition of a probe are fundamental to the specificity of that probe. The well known notion that a probe should be greater than 50% guanine and cytosine is paralleled by the need for the probe to be specific for the target sequence. Generally a probe must be a minimum of 18 bases to be highly specific for a target sequence within most genomes. The probability that the probe may hybridize to more than one location can be minimized by using software which will not only choose a probe which lacks secondary structure, but also assesses whether the sequence will appear in other known sequences from the genome under examination, or from any genome within sequence libraries (e.g., NCBI’s Genebank or EMBL database). Probe analysis software is a crucial tool for designing quality probes as well as PCR primer sets. Many good DNA analysis software programs are available and can be found by a basic internet search.
Most DNA hybridization assays still make use of target DNA transferred to a membrane by a blotting protocol. Following separation of target DNA by electrophoresis, the DNA is transferred to the membrane by one of several techniques, such as electro-, vacuum, or capillary blotting. All work well, but the method which gives fastest transfer will yield the sharpest bands since the diffusion of DNA in the gel during the transfer is minimized. The choice of membrane also impacts the quality of the assay results. The traditional medium for blotting is nitrocellulose, however, PVDF and nylon membranes can be used as well. When selecting a membrane type, realize the method used for detection must be compatible with the membrane. Many chemiluminescent substrates used with non-radioactive blotting will be incompatible with either nitrocellulose or nylon.
Though the vast majority of researchers use nitrocellulose, it tends to bind only larger DNA fragments (>300 bp). Nylon has the advantage of binding small fragments and increased durability (it is very difficult to tear). Do not discount PVDF as a membrane choice as it binds DNA well and can be used with a wide variety of chemiluminescent substrates during detection.
The hybridization process is performed in an aqueous buffer in the presence of surfactants and blocking agents. Original blotting protocols called for a sodium chloride/sodium citrate hybridization buffer which contained SDS, denatured salmon sperm DNA, Ficoll, BSA, and polyvinylpyrrolidone. Dextran sulfate can be added as an exclusion reagent (i.e., taking up space occupied by water) and thus forcing the probe closer to the membrane. Formamide can also be added as it disrupts poorly hybridized molecules and tends to prevent non-specific hybridizations. Though these solutions do work, analysis of the various buffers has shown that a good hybridization solution can actually have a simpler recipe.
One solution can be used for both prehybridization and hybridization and can be as simple as 5X SSC (diluted from 20X SSC: 0.3 M Na citrate, pH 7, 3 M NaCl), 1.0% protein blocker (either casein or BSA), 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate. This solution can be aliquoted and stored at -20°C or colder.
For blots, several different configurations can be used for hybridization. A simple method which works well involves sealing the blot in a polypropylene bag with hybridization solution. The blot is simply placed between two sheets of polypropylene and sealed around three sides (an impulse heat sealer is a needed tool). Prehybridization solution is added to the bag, bubbles are forced out the top (this takes a little practice), and the bag is sealed across the opening leaving an inch or more space above the blot. The bag is floated in a circulating water bath with the DNA side down. This helps to prevent small bubbles from interfering with probe hybridization. After one to four hours, the bag is removed, the bag is opened at the top, and the probe is added with a micropipette. Do not touch the pipette against the membrane as this will cause a significant background. Instead, tip the bag so to form a pool of solution, and then add the probe to the pool. Seal the bag, redistribute the hybridization solution, and return to the water bath overnight.
Accurate and consistent heat is crucial for specific hybridization of a probe to its complementary sequence. In practice, hybridization assays are performed in water baths, high temperature incubators, and low temperature ovens, all of which may be suitable for the application. However, a less apparent consideration is whether heat is provided constantly and evenly. Many incubators and ovens may have wide fluctuations in temperature and may possess hot/cold spots within the chamber. Often best results for membrane assays are obtained by floating the blot in a sealed plastic bag in a circulating water bath. These baths tend to provide consistency and efficient heat transfer.
A second major temperature consideration concerns the denaturing of double-stranded probes, such as those generated from random priming and nick translation, and preventing re-annealing. This is best accomplished by boiling the probe in a screw capped microfuge tube for several minutes followed by plunging the tube into a dry ice/ethanol bath or ethanol stored in a -80°C freezer. Ethanol chilled to any temperature lower than -20°C down to its freezing point (-117°C) will work, with the colder temperatures being preferable. Flash freezing will prevent re-annealing. The probe can be thawed and once liquid, added to the prehybridization solution. Often it is advisable to dilute the probe into hybridization solution before boiling so to dilute the strands and slow the rate of snap back.
Assays with clear signal and low background are dependent upon both suitable concentrations of target molecules and probe. Assays in which the target DNA concentrations are either too low or high will yield either low signal, or in the case of a blot, lack of resolution between multiple bands. Using excessive amounts of probe will cause background as well. Blots which have evenly distributed high background are caused by high probe concentration or insufficient blocking.
Solution vs. Solid Phase Assays
Whether a hybridization assay is solution based or has one immobilized strand will drastically impact the assay. The most important parameter affected is time required for hybridization. For instance, the polymerase chain reaction is, for all practical purposes, a hybridization assay which requires less than a minute for sequence specific primers to anneal to their targets. Conversely, a Southern blot requires up to 16 hours or longer for hybridization to occur. This exemplifies the difference between the kinetics of solution and solid phase hybridization. Hybridization assays performed in microtiter wells sit in the middle with a small volume of hybridization solution (e.g., 50 μl) being exposed to a large probe coated surface, i.e., up to 1 cm2. Hybridizations in this environment can be performed in 1-2 hours.