DNA concentration is a fundamental metric in molecular biology, defining the mass of genetic material per unit volume, typically expressed as nanograms per microliter (ng/µL). Accurately determining this measurement is the foundational step before initiating any experiment, such as polymerase chain reaction (PCR) or sequencing. An incorrect concentration can lead to failed reactions, wasted reagents, or unreliable data. However, concentration only tells half the story; the quality and purity of the sample are equally important factors.
Methods for Measuring DNA Concentration
The two most common laboratory techniques for measuring DNA concentration are UV spectrophotometry and fluorescence-based assays. UV spectrophotometry measures the absorbance of ultraviolet light at 260 nanometers (nm). Since nucleic acids absorb light maximally at this wavelength, the absorbance value is converted into a concentration in ng/µL. An absorbance of 1.0 at 260 nm corresponds to 50 ng/µL of double-stranded DNA.
A significant limitation is that UV spectrophotometry measures all molecules that absorb at 260 nm, including RNA and contaminants, potentially leading to overestimation. In contrast, fluorescence-based methods use specific fluorescent dyes, such as PicoGreen, that bind selectively to double-stranded DNA. A fluorometer measures the emitted light signal, which is proportional only to the functional DNA concentration. This technique is preferred for sensitive applications, like Next-Generation Sequencing, because it provides a more accurate and specific measure.
Concentration Requirements for Common Applications
The optimal DNA concentration varies significantly depending on the specific molecular application. Standard Polymerase Chain Reaction (PCR) is robust and has a broad template requirement. A typical reaction may require 1 nanogram (ng) to 1 microgram (µg) of genomic DNA, or 1 picogram (pg) to 10 ng of plasmid DNA. Adding too much template can hinder the reaction by increasing non-specific amplification or exhausting reagents prematurely.
Quantitative PCR (qPCR) is more sensitive and requires a lower input concentration, typically 0.5 ng/µL to 2 ng/µL in the final reaction mix for reliable quantification. Next-Generation Sequencing (NGS) library preparation requires the most stringent concentration control due to complex enzymatic steps. For whole-genome sequencing, the recommended input is often 100 ng to 1 µg of high-quality genomic DNA. While specialized kits allow for less, high-quality results are most reliably achieved within the manufacturer’s specified range.
The Importance of DNA Purity Ratios
While concentration measures the quantity of DNA, purity ratios measure the sample’s quality by detecting common contaminants that inhibit downstream enzymes. Spectrophotometers calculate two primary metrics: the A260/A280 ratio and the A260/A230 ratio.
The A260/A280 ratio assesses contamination by proteins and residual organic compounds, which absorb light strongly at 280 nm due to the aromatic amino acids they contain. A pure double-stranded DNA sample should have a ratio of approximately 1.8. A ratio lower than 1.7 suggests significant protein or phenol carryover, which interferes with polymerase activity and reduces enzymatic efficiency.
The A260/A230 ratio checks for contamination from chaotropic salts, such as guanidine, or organic compounds like phenol and EDTA. These reagents are frequently used during the DNA extraction process and absorb light near 230 nm. A pure DNA sample should exhibit an A260/A230 ratio between 2.0 and 2.2. Ratios significantly lower than 2.0 indicate inhibitory contaminants that compromise sensitive applications like NGS library preparation. A good DNA sample must meet the required concentration and pass both purity ratio checks.
Adjusting and Storing DNA Samples
After determining concentration and purity, the DNA sample often needs adjustment to meet experimental requirements. If the concentration is too high, the sample can be diluted using a suitable buffer, such as Tris-EDTA (TE) or molecular-grade water. If the sample is too dilute, concentration can be increased through methods like ethanol or isopropanol precipitation, where the DNA is pelleted and resuspended in a smaller volume.
For samples requiring high concentration or removal of volatile solvents, methods like speed vacuum concentration or lyophilization (freeze-drying) can be used. Proper storage is important for maintaining DNA integrity. For short-term use, samples can be stored at -20°C, but long-term preservation requires storage at -80°C. To prevent damage from repeated freezing and thawing, it is best practice to divide the sample into smaller working aliquots.