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In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance.
DNA and RNA quantification, generally referred to as nucleic acid quantification, is commonly performed to determine the average concentration of DNA or RNA in a sample prior to downstream experiments.
The real-time PCR (qPCR) technique is a powerful nucleic acid quantitation method. This is a good choice for qualitative as well as quantitative analysis of DNA because of its high sensitivity and specificity for typical molecular applications.
There are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide.
Real-time PCR offers the most sensitive detection, as low as picogram quantities of nucleic acid. Real-time PCR can accurately quantitate a subset of specific nucleic acids of interest, even in the presence of common contaminants, other nucleic acids, primers and free nucleotides.
Accurate nucleic acid quantification is a critical step in DNA and RNA sample prep workflows. Often success or failure in downstream analysis depends on whether the appropriate amount and quality of input nucleic acid is used.
NUCLEIC ACID QUANTITATION. The quantitation of nucleic acids can be accomplished either by directly converting absorbance to concentration, or by fluorescence assays. The gold standard of DNA and RNA quantitation remains UV absorbance measurements at 260 nm.
Using droplet digital PCR, our results provide strong evidence that standard curve-free nucleic acid quantification is achievable for NGS libraries and gene expression.
In this work, we introduced, analyzed, and validated hybridization probe sets with LLR and showed that they can be used for accurate quantitation of nucleic acid concentrations across many orders of magnitude.
Nucleic acid quantification encompasses key methods, including: Spectrophotometric determination of dsDNA, ssDNA, RNA at A260. Fluorometric determination of nucleic acids with fluorescent dyes, for example, PicoGreen. Determination of purity, based on A260/A280 ratios. Spectral scanning.
To check the success of your DNA extraction protocols and PCR reactions, and to determine the concentration, yield and purity of your DNA samples, you usually need to use a combination of quantification methods.
An example of the calculation involved in nucleic acid quantification when using a spectrophotometer (see Spectrophotometric measurement of DNA concentration).
Resources. Student Resource Center. Lab Essentials. Nucleic Acid Purification and Quantitation. In today’s world of nucleic acid analysis by real-time PCR and next-generation sequencing, it’s crucial to purify high-quality nucleic acids from your samples.
Quantification of DNA through the NanoDrop Spectrophotometer: Methodological Validation Using Standard Reference Material and Sprague Dawley Rat and Human DNA - PMC. As a library, NLM provides access to scientific literature.
Nucleic acid detection and quantification methods. Nucleic acid is often purified from cells as part of an ever-growing array of molecular biology methods, including sequencing and gene editing. Before they are used in downstream applications, nucleic acids are detected and quantitated using UV or fluorescence spectrophotometry.
Molecular Probes scientists have developed rapid and exceptionally sensitive fluorescence-based assays for quantitation of nucleic acids in solution. The Quant-iT Assay Kits represent the most advanced quantitation systems for DNA, RNA or protein samples.
Here we develop a PCR-based molecular barcoding NGS approach, quantitative amplicon sequencing (QASeq), for accurate absolute quantitation scalable to over 200 quantitation modules.
Nucleic acid quantification determines the concentration and quality of nucleic acids prior to proceeding downstream. If you run a gene expression experiment and do not get results, quantifying the RNA in your sample is a useful troubleshooting experiment. Fluorescence and spectrophotometry are two commonly used quantification methods.
A real-time polymerase chain reaction maps the concentration of a target nucleic acid sequence log-linearly into cycle threshold to enable quantitation with a wide dynamic range but suffers from enzymatic biases.
Large-scale regular nucleic acid testing (NAT) such as Regular-1/7, played a crucial role in the successful and sustained containment of COVID-19 in China, enabling rapid case detection in low ...