The Polymerase Chain Reaction (PCR) is a widely used laboratory technique that allows scientists to create millions to billions of copies of a specific DNA segment from a small initial sample. For successful PCR amplification, precision in experimental setup is important, and the amount of starting material, known as template DNA, plays a significant role in the outcome.
The Right Amount of DNA
The quantity of template DNA needed for a PCR reaction is not a fixed value, but rather falls within a recommended range depending on the type of DNA. For standard PCR, general guidelines suggest using between 1 to 100 nanograms (ng) of genomic DNA. Some protocols may even recommend up to 500 ng of human genomic DNA for a 50 microliter (µL) reaction. In contrast, less complex DNA, such as plasmid DNA, typically requires a much smaller amount, often ranging from 0.1 to 10 ng, or even as low as 1 picogram (pg) per reaction. These quantities serve as starting points, and the optimal amount can vary, requiring adjustment for specific experiments.
Factors Affecting DNA Needs
The optimal amount of template DNA is influenced by several factors. The purity of the DNA sample is a significant consideration, as contaminants co-purified with DNA can inhibit the PCR reaction. The source of the DNA also matters; bacterial DNA, originating from organisms with smaller genomes, generally requires less template compared to human or other eukaryotic genomic DNA. This relates to genome complexity, where smaller genomes mean a higher copy number of the target sequence per nanogram of DNA.
The specific type of PCR also alters DNA requirements. Quantitative PCR (qPCR), for example, is highly sensitive and can detect target DNA with as few as 1 to 10 copies.
The length of the target DNA segment, known as the amplicon, also affects optimal input. Shorter amplicons (e.g., 75-150 bp for qPCR or 200-1000 bp for standard PCR) amplify more efficiently, potentially requiring less template. Longer sequences may necessitate adjustments in enzyme and reaction conditions. The type of DNA polymerase used can also influence the optimal amount of input DNA.
Measuring DNA for PCR
Accurately determining DNA concentration is important before PCR to ensure consistent results. Two common methods for DNA quantification are spectrophotometry and fluorometry. Spectrophotometry, often using instruments like a NanoDrop, measures ultraviolet light absorbance at 260 nanometers (nm), a wavelength where DNA absorbs strongly. While simple to use, spectrophotometry does not distinguish between DNA and RNA, meaning RNA contamination can lead to an overestimation of DNA concentration.
Fluorometry uses fluorescent dyes that bind specifically to double-stranded DNA. This method is more sensitive than spectrophotometry, detecting DNA at picogram levels and providing more accurate measurements because dyes only fluoresce when bound to target DNA. Therefore, fluorometry is often preferred for low concentration samples or when high precision is required. Agarose gel electrophoresis can also offer a visual, rough estimate of DNA quantity and indicate degradation or contamination.
Impact of DNA Quantity
Using an incorrect amount of template DNA can lead to undesirable PCR outcomes. Too much DNA can cause primers to bind to unintended sites, resulting in non-specific amplification and unwanted DNA fragments. Excess DNA can also promote primer-dimer formation, short DNA fragments created when primers bind to each other instead of target DNA. High DNA concentrations can also inhibit the PCR reaction, possibly by binding essential cofactors like magnesium ions or introducing inhibitory contaminants.
Conversely, using too little template DNA can also compromise PCR success. Insufficient DNA may lead to no amplification or only very weak, undetectable signals. This can result in inconsistent or irreproducible results, especially with very low target DNA copy numbers. Ultimately, achieving the optimal balance in template DNA quantity is important for specific, strong, and reliable PCR amplification.