Molecular biology reactions require very precise amounts of nucleic acids for optimal performance. Too little or too much nucleic acid can severely impact the final assay results, so quantification should be a standard procedure following purification to ensure successful reaction outcomes.
Incorrect assumptions regarding sample quantity can occur for a variety of reasons, so selecting an appropriate method that provides accurate quantification is critical.
Back to topInsight offered by quantification
Measuring the concentration of nucleic acid samples is a key QC step to determine the fit and amount of nucleic acid available for further processing.
Back to topImpact of quantity on downstream steps
Overestimating the DNA concentration leads to too little input DNA in molecular reactions, and in the case of PCR, results in weak amplification and weak signal strength. PCR yield is also reduced by large amounts of RNA in the DNA template, or by inhibitors such as residual EDTA or other negatively charged ions that chelate Mg2+
(see also Purity
Underestimation of DNA concentration leads to too high an amount of input DNA. In PCR, too much template DNA causes a variety of issues that can negatively impact the reaction performance:
Back to topTechnologies for assessing nucleic acid quantity
- Unfavorable ratios of primer, template and enzyme and suboptimal reaction conditions
- False priming and generation of non-specific products
- Obstructed diffusion of Taq polymerase molecules in the reaction
- Unnecessary increase of inhibitors in downstream assays
- False-negative results
Nucleic acid quantification is commonly performed using a spectrophotometer to analyze spectral absorbance or by measuring the fluorescence intensity of the sample in the presence of specific DNA- or RNA-binding dyes.
Fluorescence-based methods are generally more sensitive than absorbance-based methods, but they require additional steps for incubating samples with the fluorescent dyes and creating standard curves with known quantities of nucleic acid. A disadvantage of fluorescence measurements is that they provide no information regarding sample impurities. Furthermore, since the dye binds specifically to either double- or single-stranded DNA or RNA, the concentration calculated using these methods can lead to incorrect assumptions regarding total nucleic acids in the samples.
Spectral absorbance methods involve measuring the sample's absorbance of ultraviolet (UV) light using a spectrophotometer and can provide important information regarding nucleic acid quantity and purity. Different molecules such as nucleic acids, proteins and chemical contaminants absorb light in their own particular pattern; for example, nucleic acids have a peak of absorbance at 260 nm. By measuring the amount of light absorbed at a defined wavelength, the concentration of the molecules of interest can be calculated.
Every nucleic acid quantification method has its own advantages and limitations, and some systems might be preferable for particular sample types. Studies suggest that nucleic acid quantification using UV absorbance shows the highest concordance with reference samples and is influenced less by the degree of fragmentation and nucleic acid quality (2). Next-generation spectrophotometry applies spectral content profiling to measure the whole nucleic acid profile and can differentiate between DNA, RNA and other absorbing contaminants.
Back to topQIAGEN solutions for nucleic acid quantification
The QIAxpert system
is the next generation in UV/Vis spectrophotometry, using proprietary spectral content analysis to unmix the spectra and fit reference sample and buffer components to correctly discriminate between DNA, RNA and impurities.
Find out more about other important parameters for sample QC:
Back to topReferences
- Haque, K.A., Pfeiffer, R.M., Beerman, M.B., Chanock, S.J., and Bergen, A.W. 2003. Performance of high-throughput DNA quantification methods. BMC Biotechnology, 3,20. DOI: 10.1186/1472-6750-3-20.
Sedlackova, T., Repiska, G., Celec, P., Szemes, T., and Minarik, G. 2013. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biological Procedures Online, 15,5. http://doi.org/10.1186/1480-9222-15-5.
- Schade, C. 2014. Quality Control: An Important Success Factor in Nucleic Acid-Based Analysis. American Laboratory Articles Website. www.americanlaboratory.com/914-Application-Notes/158842-Quality-Control-An-Important-Success-Factor-in-Nucleic-Acid-Based-Analysis/.