Advances in Nucleic Acid Detection

An Overview of Current Tools for DNA and RNA Quantification

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When discussing nucleic acid detection, there are two components to keep in mind: the detector and the transducer. The detector is the part which recognizes the nucleic acid and interacts directly with it. The transducer is the part which then generates a quantifiable signal from that interaction. Take, for example, the well-developed DNA detection method called fluorescence in situ hybridization (FISH).

FISH was first developed in the 1980s as a method to detect specific sequences of DNA on chromosomes. It works by designing a single stranded DNA (ssDNA) probe that is complementary to the target sequence. The probe needs to have enough base pairs to bind specifically to the target sequence while small enough to not interfere with hybridization. Once a probe, or detector, is designed, it is then tagged with a fluorophore which acts as a signal transducer and allows for sequence detection events to be quantified. With the probe prepared, the sample DNA is denatured, and the probe is added. As the DNA anneals, hybridization will occur, wherein the probe and the target ssDNA will combine to reform double stranded DNA (dsDNA). Then the probe, and consequently the target DNA sequence, can be detected using standard instrumentation, such as fluorescent microscopy.

While an elegant method, several considerations have to be made when using FISH and similar hybridization procedures. For example, competition between probe and sample DNA will reduce successful hybridization during annealing, leading to dsDNA comprised only of sample ssDNA. The exclusion of the probe in dsDNA will have a significant impact on signal quality. Furthermore, FISH does not reflect live cell conditions. DNA is denatured at temperatures well above that of cellular environments, and in fact, the very denaturing requirement itself poorly reflects actual cell conditions. Most DNA in cells persists as dsDNA rather than ssDNA.

Aside from cellular conditions, there are practical issues to consider. For example, FISH is a very time consuming procedure, requiring approximately 12 hours per assay. Often times, researchers simply want a quick assay to quantify DNA, without necessarily targeting specific sequences. For these types of applications, a vast array of probes has been developed that not only offer convenience of use but also detection of dsDNA without extreme denaturing procedures.

dsDNA Detection

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If specific sequence detection is not required, then there are many chemical probes available for detection of dsDNA. One of the earliest was 4',6-diamidino-2-phenylindole (DAPI).

DAPI was first developed in 1971, and it is still used extensively in fluorescence microscopy. It acts by binding to AT rich regions of dsDNA. Once bound to dsDNA, it will emit a strong signal at 461 nm when excited by an ultraviolet light source (maximal absorption at 358 nm). Because DAPI is cell permeable, it can theoretically be used to stain both fixed and live cells. It is, however, worth noting that DAPI is less permeable in live cells and thus stains much more poorly than in fixed cells.

Another commonly used chemical probe is the bisbenzimides family. Of these, the Hoechst stains are the most well established. Like DAPI, the Hoechst stains will bind to AT rich regions in dsDNA, localizing in the minor groove. The Hoechst stains have two significant advantages over DAPI, however. First, the Hoechst dyes have an extra ethyl group in their chemical structure, which makes these compounds more hydrophobic. This allows for easier access through the cell membrane of live cells and, consequently, better staining. Second, the Hoechst stains tend to be less toxic, which minimizes their impact on cellular function. This once again benefits the staining of live cells. Of the Hoechst stains, Hoechst 33258 and Hoechst 33342 are the most commonly used. These two dyes are similar in that they both excite at around 350 nm and emit a blue/ cyan fluorescent light with a maximum emission at about 460 nm. Because of their spectra, they tend to act as a good replacement for DAPI. More recently, there has been intense focus on developing probes that have greater sensitivity and selectivity for dsDNA. In particular, it is worth mentioning the development of Helixyte™ Green. Like DAPI and the Hoechst probes, Helixyte™ Green binds to the minor groove of DNA. However, it has significant improvements over these first-generation dsDNA dyes. First, Helixtye™ Green is significantly more selective. Unlike DAPI, it has very minimal binding to RNA. And in comparison to Hoechst 33258, Helixyte™ Green also shows improved binding to homopolymers, regions of dsDNA which have repeating base pairs (eg. AAAA, TTT ). In terms of sensitivity, Helixyte™ Green is purportedly one of the most sensitive probes for quantifying dsDNA in solution. According to its specifications, Helixyte™ Green will selectively detect as little as 25 pg/mL dsDNA even in the presence of ssDNA, RNA and free nucleotides. This makes it roughly 400 times more sensitive than Hoechst 33258 under the same dye concentrations. Finally, Helixyte™ Green is reported to have linearity across three orders of magnitude. This is in reference to the linearity of dilution assessment. In short, it means that assay functionality does not deviate significantly under different analyte dilutions. This translates into a robust assay with high precision across a wide range of experimental conditions and allows for quantification of DNA from a variety of sources including genomic DNA, viral DNA, miniprep DNA and PCR amplification products.

RNA Detection

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With clear advances in dsDNA detection, it is worth mentioning that there have been significant strides in RNA detection as well. In particular, StrandBrite™ Green has generated impressive results. StrandBrite™ Green is a fluorescent probe that is capable of detecting as little as 5 ng/mL of RNA in assay solution through use of instrumentation such as fluorescence microplate readers. To determine RNA selectivity, a standard DNase and RNase digest test was conducted. After DNase digestion, it was found that fixed cells stained with StrandBrite™ Green did not experience a significant change in initial fluorescence intensity. On the other hand, after RNase digestion, there was an immediate and dramatic decrease of initial fluorescence signal. Furthermore, short exposure of live cells to dantinomycin caused dose-dependent detection of inhibited RNA synthesis during the 6 hours after drug removal. All of these results strongly support the claim that fluorescence signals derived from StrandBrite™ Green are a result of specific interactions between the probe and RNA in sample cells. Due to its excellent cell permeability and spectral properties, StrandBrite™ Green has been successfully used for flow cytometry-based RNA analysis and fluorescence microscopy in live cells. It is well excited by 488 nm argon-ion lasers and uses the standard FITC channel emission filter, making it a convenient and valuable tool for the quantification of RNA.