The Targeted Restriction-site Amplification Polymorphism (TRAP) assay is a molecular biology method used to generate a unique genetic fingerprint for an organism. This Polymerase Chain Reaction (PCR)-based technique allows researchers to quickly examine genetic differences, known as polymorphisms, across multiple samples simultaneously. TRAP is especially valuable for organisms whose complete genetic blueprint is not fully known, offering a means to analyze specific gene regions without extensive prior sequence information. The assay serves as an efficient tool for comparative analysis, enabling the detection of variation in both coding and non-coding regions of the genome, which is important for understanding genetic diversity and gene function.
Defining the Targeted Restriction-site Amplification Polymorphism Assay
The acronym TRAP stands for Target Region Amplification Polymorphism, a technique developed to overcome limitations found in earlier random molecular markers. This method combines sequence-specific knowledge with an arbitrary amplification approach to focus on functional parts of the genome. This provides a reliable and reproducible way to target genes of interest and compare their variants across different individuals or strains.
TRAP achieves this focus through the specialized design of two distinct primers used in the reaction. The first is a fixed primer, typically derived from a known gene sequence, such as an Expressed Sequence Tag (EST) or other functional gene data. This fixed primer anchors the amplification process to a specific region of the genome.
The second component is an arbitrary primer, which contains a core region that is either rich in Adenine and Thymine (AT) or Guanine and Cytosine (GC) nucleotides. This arbitrary primer is designed to bind semi-randomly near the fixed primer site, often targeting an adjacent intron or exon. The combination of one highly specific primer and one semi-random primer ensures that the resulting amplified fragments are clustered around the targeted gene region.
Unlike standard PCR, which uses two highly specific primers to amplify a single, known fragment, the TRAP assay uses these two distinct primer types simultaneously to generate multiple fragments in a single reaction. This multiplexing capability generates an informative banding pattern that reflects genetic variations in the targeted region. The name sometimes includes “Restriction-site” because the resulting polymorphisms mimic the kind of variation revealed by techniques that rely on restriction enzymes, even though TRAP itself does not typically use them for fragment generation.
The Step-by-Step Mechanism of TRAP
The TRAP mechanism is a modified PCR process that relies on a differential annealing temperature strategy across its thermal cycling phases. The reaction begins with the preparation of the DNA template, which is extracted from the organism being studied, such as plant tissue. This template DNA is then mixed with the two specialized primers, a DNA polymerase enzyme, and free nucleotides for synthesis.
The first phase involves a few initial cycles, typically five, executed at a low annealing temperature, such as 35°C. At this low stringency, the arbitrary primer can bind to multiple locations on the DNA template that have only partial sequence complementarity, including sites near where the fixed primer has already annealed. This initial, less-specific binding allows the arbitrary primer to “prime” the synthesis of new DNA strands from several starting points within the target region.
Following these initial cycles, the reaction shifts into the main amplification phase, consisting of approximately 35 cycles at a higher, more stringent annealing temperature, often around 50°C. This increase in temperature ensures that only the most complementary primer-template pairs remain stably bound. The fragments initiated in the first phase, which now contain both the fixed and arbitrary primer sequences at their ends, are efficiently and exponentially amplified. The fixed primer provides the precise starting point, while the arbitrary primer provides the end point. This two-step cycling process enables the TRAP assay to generate a set of reproducible, gene-associated DNA fragments.
Interpreting Banding Patterns and Polymorphisms
The final products of the TRAP assay are separated and visualized, typically using high-resolution electrophoresis on a polyacrylamide gel. This separation process arranges the amplified DNA fragments by size, creating a distinct pattern of bands for each sample. A single TRAP reaction can yield numerous fragments, often generating 30 to 50 distinct bands ranging in size from approximately 50 to 900 base pairs.
Each visible band represents a specific DNA fragment successfully amplified between the fixed primer and one of the arbitrary primer’s binding sites. Researchers analyze these patterns by scoring the presence or absence of a band at a specific position for each sample. Bands that are present in one sample but missing in another indicate a genetic difference, which is termed a polymorphism.
This difference in banding reflects a variation in the underlying DNA sequence of the targeted region between the two samples. For example, a mutation or small insertion/deletion event might have occurred in one sample, preventing the arbitrary primer from binding and thus eliminating the amplification of that specific fragment. Conversely, a new binding site might be created, resulting in a new band. The overall comparison of these banding patterns allows for the calculation of genetic similarity or distance between the tested individuals.
Specific Applications in Molecular Biology Research
The TRAP assay is used in comparative genomics to distinguish between closely related species or to assess genetic diversity within a large population. The technique has been successfully used to characterize different varieties of crops such as sweet sorghum, sugarcane, and Malus genotypes.
The assay is also used for differential display, which is the process of identifying genes that are expressed under one condition but not another, such as during a stress response or disease. By designing fixed primers based on genes suspected of being involved in a particular pathway, researchers can screen for polymorphisms that correlate with a specific trait. This allows for the rapid identification of genetic markers associated with important characteristics.
A primary application is in marker-assisted selection, particularly in plant and animal breeding programs. TRAP markers allow breeders to quickly screen large numbers of offspring for the presence of a desired gene, such as one conferring disease resistance, without having to wait for the organism to mature and display the physical trait. This speeds up the breeding process considerably and highlights the technique’s value when working with organisms for which complete genomic sequence information is unavailable.