Multiple Displacement Amplification (MDA) is a highly efficient technique for generating large quantities of DNA from minute samples, a process known as whole-genome amplification (WGA). The method is non-PCR based and operates under isothermal conditions, meaning it maintains a constant temperature throughout the reaction. MDA’s primary purpose is to overcome the limitations of scarce starting material, such as DNA from a single cell or trace forensic evidence. This technique allows researchers to amplify an entire genome thousands of times over, producing microgram amounts of DNA suitable for extensive genomic analysis.
Core Components: The Enzyme and Primer Types
Multiple Displacement Amplification relies on a specialized DNA polymerase and specific oligonucleotide primers. The central enzyme is bacteriophage Phi29 DNA Polymerase, which originates from a virus that infects bacteria. This enzyme possesses two unique properties enabling the MDA process: extremely high processivity and inherent strand displacement activity. Processivity refers to the enzyme’s ability to remain attached to the DNA template and continuously synthesize long stretches of DNA, often exceeding 70 kilobases, without detaching.
Strand displacement means that as the enzyme synthesizes a new DNA strand, it pushes aside any existing complementary strand encountered on the template without stopping. This activity differs fundamentally from polymerases used in traditional methods like PCR, which require high heat to separate double-stranded DNA. This continuous synthesis facilitated by strand displacement allows the entire MDA reaction to proceed at a low, constant temperature, typically around 30°C.
DNA synthesis depends on random hexamer primers, which are short, 6-base DNA sequences. These hexamers are designed to anneal randomly and simultaneously to countless sites across the template DNA genome. Their non-specific binding ensures the amplification process starts everywhere on the genome at once, leading to comprehensive coverage. The hexamers create the starting points for the Phi29 polymerase, allowing whole-genome amplification to commence in an unbiased manner.
The Hyper-Branching Amplification Mechanism
The process begins with a brief preliminary step, often involving a mild alkaline treatment or low heat, to slightly denature the double-stranded DNA template. This step helps to separate the two DNA strands, making them accessible to the random hexamer primers. Once the template strands are separated, the random hexamer primers quickly anneal to the numerous complementary sequences scattered throughout the single-stranded template.
The Phi29 DNA Polymerase binds to the 3’ end of each annealed hexamer and initiates DNA synthesis, extending the primer. As the polymerase moves along the template, it encounters previously synthesized DNA strands. Instead of stopping, it uses its strand displacement activity to push the existing strand off the template, generating a new single-stranded DNA product that becomes available as a new template.
The newly displaced single strand is free, and its exposed sequence allows more random hexamer primers to anneal. This secondary priming event immediately initiates further DNA synthesis, which leads to more strand displacement and the generation of additional single-stranded templates.
This mechanism is termed “hyper-branching” because the amplification signal propagates through a cascade of continuous synthesis and displacement events. The entire MDA reaction occurs at a single, consistent temperature, unlike the cyclical changes required for PCR. The result of this process is a massive, branched concatemeric structure that contains thousands of copies of the original genomic DNA.
Key Applications and Product Characteristics
Multiple Displacement Amplification is frequently utilized in fields where the starting biological material is scarce. Primary applications include:
- Single-cell genomics, where researchers amplify minute DNA content from a single cell (e.g., in cancer research or developmental biology).
- Metagenomics, enabling the amplification of trace amounts of DNA from environmental samples or uncultured microorganisms.
- Forensic science, generating sufficient DNA for analysis from trace evidence like hair or saliva.
The amplified DNA product generated by MDA is highly suitable for downstream analysis. The high processivity of the Phi29 polymerase results in high molecular weight DNA, with fragments often exceeding 10 kilobases and occasionally reaching up to 100 kilobases. This production of long DNA molecules is beneficial for techniques requiring intact segments, such as sequencing library preparations or restriction fragment length polymorphism analysis.
MDA is known to produce a high yield of DNA, routinely generating tens of micrograms of product from picogram amounts of starting material. The amplification exhibits relatively uniform coverage across the entire genome, meaning that most regions of the genome are represented in the final product without significant bias. This high fidelity and low bias allows for accurate downstream genetic studies, including single-nucleotide polymorphism (SNP) analysis and whole-genome sequencing.