Overview: Hybridization Buffers and Solutions
Nucleic Acid Hybridization
Nucleic Acid Hybridization is the process in which single stranded nucleic acid molecules (DNA or RNA) bind to their complementary sequences to form double stranded molecules [1]. Complimentary base pairing is based on the Watson-Crick base pairing principle, in which purine (Adenine and Guanine) and pyrimidine (Cytosine and Thymine in DNA, and Uracil in RNA) bases bind together [2]. This process is completely natural, and is an essential component of DNA replication, gene regulation and cell cycle. The results of nucleic acid hybridization are heavily dependent on the type, extent, and genetic location of the hybridization, and is known to lead to genetic recombination, which can create new combinations of alleles and expression. This often occurs during reproduction of many organisms, leading to the genetic diversification of subsequent generations. Due to its unique nature, genetic expression due to nucleic acid hybridization holds a large influence in the research and development of gene therapies, making this process extremely valuable and a hot spot in drug development, and therapeutic research.
The Hybridization Process
The process of hybridization typically involves a denaturation step to break the bonds of a double stranded nucleic acid, so each strand is capable of binding to an equally denatured probe [3]. Denaturation can occur using heat, or in some cases by using a chemical denaturant or enzyme. A probe in this case is a single-stranded nucleic acid with a radioactive isotope or fluorescent dye label, for later analytical purposes. After denaturation, hybridization of the probe and single stranded nucleic acids can occur in the presence of a hybridization buffer, which prevents strands from re-annealing or nonspecific binding. This way, the probe can bind correctly to the DNA strand of interest, and expression can occur. By manipulating the nucleic acid probe, scientists can transfect sample cells, and track biomarkers linked to the associated gene expression. This process is very specific, and must utilize the appropriate enzymes, buffers, solutions, and media unique to the cells of interest [4].
Image 1: The hybridization process is a biologically conserved mechanism that allows for the diversification of genetic expression. Here, a denatured DNA probe binds to a denatured DNA strand through the process of annealing. This occurs in the presence of a hybridization buffer.
Hybridization Buffers:
Hybridization buffers are solutions that can be used to facilitate the binding of nucleic acid probes to target sequences in tissue samples, on the membranes, or microarrays. This process is a key component in several experiments including but not limited to:
- In situ hybridization (ISH) to localize and quantify DNA or RNA in a variety of fixed tissues and cells preparations in order to obtain temporal and spatial information about gene expression using chromogenic detection.
- Fluorescent in situ hybridization (FISH) to detect specific complementary sequences on a chromosome by using fluorescent-labeled DNA probes which hybridizes with a particular portion of the genome.
- DNA microarray analysis to measure the expression levels of thousands of genes simultaneously using probes.
- Southern Blotting to identify specific DNA fragments from a mixture of DNA fragments that have been separated by gel electrophoresis using probes.
- Northern Blotting to identify specific RNA molecules from a mixture of RNA fragments that have been separated by gel electrophoresis using probes.
- Polymerase Chain Reaction (PCR) to amplify specific DNA sequences through repeated cycles of denaturation, annealing, and extension.
- Next-Generation Sequencing (NGS) to profile or selectively sequence specific regions of interest in the genome, enabling high-throughput sequencing.
The composition of Hybridization buffers can vary depending upon their applications, however the components within this buffer type must meet specific criteria. First, they must lower the melting temperature of nucleic acids of interest, allowing for the separation and re-annealing without fail. They must result in stabilizing the nucleic acid structure to allow it to withstand hybridization kinetics, reduce nucleic acid aggregation by reducing surface tension, and prevent non-specific binding. To meet all of these specifications, hybridization buffers are often made with the following chemical types.
- Formamide: Formamide can help to lower the melting temperature of nucleic acid strands and reduce background noise during hybridization and later expression.
- Buffer: A buffer is a solution that can help regulate the pH of a solution. In the case of hybridization buffers, buffers such as Tris-acetate-EDTA (TAE), or Saline-Tris-EDTA (STE) or Saline-sodium citrate (SSC) can be used.
- Salt: Salts provide an ion source in solution capable of stabilizing nucleic acid structures, which also support hybridization kinetics. Salt concentrations vary on the nucleic acid in question. Types of salts often used in hybridization buffers often include Sodium Chloride (NaCl) or Sodium Acetate.
- Detergent: A detergent is a surfactant that reduces surface tension and prevents nucleic acid aggregation. It can also remove excess probe while reducing non-specific binding. Types of detergents generally used are Sodium Dodecyl Sulfate (SDS), Tween-20, or Triton X-100.
- Blocking Agent: A blocking agent is a substance used for minimizing non-specific binding and unwanted background signals, by binding to the membrane or surfaces used during hybridization process. Common blocking agents used are Bovine Serum Albumin (BSA), Salmon Sperm DNA, calf thymus DNA or yeast tRNA.
- Labeling Agent: A labeling agent is a molecule attached to the probe used and allows the user to detect hybrid complexes. This can be a radioactive agent like 32P, 35S or 3H, an enzymatic agent like alkaline phosphatase or horseradish peroxidase, or a fluorescent agent like fluorescein or rhodamine.
Hybridization Buffers at Boston BioProducts
Every Hybridization Buffer is unique to the cell type used and the experimental application. Select the appropriate Hybridization Buffers from the catalog or design your optimal formulation with custom manufacturing options at Boston BioProducts.
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References:
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- Cornish, E. C., Beckham, S. A., & Maddox, J. F. (1998). Southern hybridization revisited; probe/target DNA interaction is affected by the choice of hybridization buffer. Biotechniques, 25(6), 948-954.
- Bolli, M., Trafelet, H. U., & Leumann, C. (1996). Watson-Crick base-pairing properties of bicyclo-DNA.
- Wetmur, J. G. (1991). DNA probes: Applications of the principles of nucleic acid hybridization. Critical Reviews in Biochemistry and Molecular Biology, 26(3-4), 227-259
- Kennell, D. E. (1971). Principles and practices of nucleic acid hybridization. Progress in Nucleic Acid Research and Molecular Biology, 11, 259-301.