In modern molecular biology research, nucleic acid quantification is an essential step. Accurate quantification of nucleic acids is crucial for ensuring the reliability and reproducibility of experimental results. Knowing the precise concentration of nucleic acids in a sample is the key to success in PCR, gene sequencing, or any other molecular biology experiments.
The main importance of nucleic acid quantification is reflected in the optimization of experimental conditions, the evaluation of sample quality, the assurance of the success of downstream applications, and the saving of time and resources. Many experiments require specific concentrations of nucleic acid input, and accurate quantification helps researchers optimize experimental conditions. Through quantification, we can assess the efficiency of DNA extraction and purification, and detect potential contamination. In applications such as NGS (Next Generation Sequencing), accurate nucleic acid quantification is critical for library preparation. Furthermore, accurate quantification can prevent experimental failures due to inappropriate nucleic acid concentration and save valuable time and resources.
Given the importance of nucleic acid quantification, choosing the appropriate quantification method becomes particularly crucial. Different methods have their own advantages and disadvantages, suitable for a range of different experimental scenarios.
Researchers have a choice of several methods to choose from when quantifying nucleic acids, each tailored to specific experimental needs and based on different principles. The most common methods include UV-Vis spectrophotometry, fluorimetry, real-time quantitative PCR (qPCR), gel electrophoresis and capillary electrophoresis. These techniques vary in their complexity and precision, from the quick and straightforward UV-Vis method to the highly accurate qPCR, offering researchers a range of options to suit their requirements.
UV-Vis spectrophotometry is one of the most commonly used nucleic acid quantification methods, known for its simplicity and speed. This method is based on the characteristic of nucleic acids having maximum absorption at a wavelength of 260 nm. By measuring the absorbance of a sample at this wavelength, the concentration of nucleic acids can be calculated. The steps include calibrating the instrument with buffer, measuring sample absorbance, and using a formula to calculate the nucleic acid concentration.
The advantages of the UV-Vis method lie in its speed and simplicity, no special reagents are needed, and it can assess sample purity. However, there are some limitations, the most serious being that it cannot distinguish between DNA and RNA, it is not sensitive enough for low samples of low concentration and it is susceptible to contaminant interference. In routine concentration detection after plasmid DNA extraction or genomic DNA extraction, the UV-Vis method is the preferred choice. The latest trend, which greatly improves work efficiency, is the development of micro-volume spectrophotometers, which allow measurements with only 1-2 μL of sample.
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Fluorometry offers highly sensitive and specific nucleic acid quantification making it especially suitable for measuring low concentration samples. Fluorescent dyes that specifically bind to nucleic acid molecules are used and measurement of fluorescence intensity allows accurate quantification of nucleic acids. Operational steps include preparing a standard curve, mixing samples with fluorescent dyes, measuring fluorescence intensity, and calculating nucleic acid concentration based on the standard curve.
The advantages of fluorometry include high sensitivity, high specificity, and minimal influence from contaminants. However, it requires special fluorescent dyes and instruments, which are relatively costly, and the need to create a standard curve. Fluorometry is the preferred method used in NGS library quantification and the quantification of low concentration samples such as cfDNA. The latest trend is the development of new fluorescent dyes that improve the sensitivity and specificity of detection.
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qPCR is a highly sensitive and specific nucleic acid quantification method that combines PCR amplification with fluorescence detection. It quantifies the initial sample nucleic acid amount by real-time monitoring of the intensity of the fluorescence signal generated during the PCR process. Operational steps include the design of specific primers and probes, the preparation of reaction reagents, running the qPCR program, and analyzing the amplification curves and Ct values.
The advantages of qPCR lie in its extremely high sensitivity, high sequence specificity, and the ability to perform quantitative and qualitative analysis simultaneously. However, it requires specialized equipment and reagents, is relatively costly, and operation is complex and time-consuming. qPCR is the preferred method for the detection of gene expression levels, viral load quantification, or the quantification of extremely low concentrations of nucleic acids. It is also the most reliable quantification method for degraded DNA in FFPE samples.
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Gel electrophoresis is a visual nucleic acid quantification method, particularly suitable for assessing the integrity and size of nucleic acids. It is based on the principle that the migration speed of nucleic acids in an electric field is inversely proportional to their molecular weight. Semi-quantitative analysis is done by comparing the brightness of samples with that of standards of known nucleic acid concentration.
The advantages of gel electrophoresis lie in an ability to simultaneously assess the concentration, size, and integrity of nucleic acid in a sample, the provision of intuitive visualization, and relatively inexpensive equipment. However, analyses are only performed semi-quantitative, sensitivity is low, and the use of toxic dyes may pose safety concerns. Gel electrophoresis is very useful for checking PCR products, assessing nucleic acid integrity, or the rough estimation of nucleic acid concentration.
Capillary electrophoresis is a highly automated method that can simultaneously analyze nucleic acid concentration and size. It combines electrophoretic separation and fluorescence detection technologies. The nucleic acids are analyzed by high-resolution separation in fine capillaries. Operational steps include sample preparation and loading, automatic capillary electrophoresis separation, fluorescence detection, and data analysis and report generation.
The advantages of capillary electrophoresis are high throughput, a high degree of automation, and the ability to measure both nucleic acid concentration and fragment size at the same time. However, the equipment is expensive, sample preparation can be complex, and highly skilled operators are needed. Capillary electrophoresis shows unique advantages in NGS library quality control, nucleic acid fragment analysis, and large-scale sample screening.
Choosing the appropriate nucleic acid quantification method is crucial for experimental success. The main factors to consider are sample type and concentration, purity, specificity requirements, downstream applications, and time and cost. UV-Vis spectrometry is suitable for highly concentrated samples (>10 ng/μL). Fluorometry or qPCR are needed for low concentration samples (<10 ng/μL) and in situations requiring distinction between DNA/RNA, or the detection of specific sequences. Fluorometry or qPCR are typically used in NGS library quantification, while qPCR is most reliable for FFPE samples.
|
Method |
Sensitivity Range |
Main Advantages |
Main Limitations |
Applicable Scenarios |
|
2-5 ng/μL |
Fast, simple, no special reagents required. |
Cannot distinguish DNA/RNA, susceptible to contaminants. |
Medium to high concentration pure samples, rapid detection. |
|
|
0.1-0.5 ng/μL |
High sensitivity, can distinguish DNA/RNA. |
Requires standard curve, higher reagent cost. |
Low sample concentration and where high accuracy is needed. |
|
|
<0.1 ng/μL |
Extremely high sensitivity, sequence specificity. |
Expensive equipment and reagents, time-consuming. |
Extremely low concentrations, specific sequence detection. |
|
|
Gel Electrophoresis |
1-5 ng/band |
Visualizes DNA size and integrity. |
Semi-quantitative, low sensitivity. |
Nucleic acid integrity check, PCR product verification. |
|
Capillary Electrophoresis |
0.1-0.5 ng/μL |
High throughput, automated. |
Expensive equipment, complex sample preparation. |
Large-scale sample analysis, NGS library quantification. |
It's worth noting that in daily laboratory work, UV-Vis spectrophotometry and fluorometry are the two most commonly used methods due to their convenience and wide applicability. The UV-Vis method is suitable for the rapid detection of medium to high concentration pure DNA samples, while fluorometry excels in handling samples of low concentration or when high accuracy is required.
Generally, results from fluorometry and qPCR are more reliable, especially for low concentration samples. UV-Vis methods may overestimate concentrations due to contaminants. It's recommended to cross-validate important samples using multiple methods.
Use fluorometry or qPCR, as these methods are more sensitive to low DNA concentrations. Increase the number of replicate measurements, and consider using carrier DNA to reduce sample loss. Ensure the use of low-adsorption centrifuge tubes and pipette tips.
qPCR or specific fluorometry methods (such as Qubit) are most suitable for NGS library quantification. qPCR can specifically detect library molecules with adapters, while fluorometry provides accurate total DNA concentration.
For FFPE samples, qPCR is the most reliable method. It can specifically detect intact nucleic acid fragments, and avoid interference from degraded nucleic acids in the measurement.
Nucleic acid quantification is a critical step in molecular biology research. Each method has its unique advantages and suitable applications. Selecting an appropriate method requires consideration of multiple factors. In routine laboratory work, UV-Vis spectroscopy and fluorometry remain the most commonly used techniques. The demand for more sensitive and precise DNA/RNA quantification methods has resulted in the rapid development in fields such as single-cell analysis, liquid biopsy, and gene therapy. In the future, we may witness innovative approaches combining nanotechnology and artificial intelligence, potentially revolutionizing our understanding of nucleic acid quantification. Continuous improvement of nucleic acid quantification techniques, not only enhance experimental accuracy and efficiency but also open up new possibilities for life science research.