METHODS OF NUCLEIC ACID DETECTION

Nucleic acid molecules like deoxyribonucleic acids (DNA), ribonucleic acids (RNA) are basic, essential and primary molecules for all molecular biology related research. Before discussing the common methods to detect nucleic acids let us we learn about the isolation of pure form of nucleic acid.

Preparation Step:

First important step is to establish if a given sample contains DNA or RNA and whether it is in pure form, since many samples will contain both species as well as other contaminants such as cellular proteins. Irrespective of the method applied for detection, the optimal nucleic acid isolation protocol must provide

a)    Reproducible result

b)    No degradation of the sample and

c)    Safer handling.

In deciding what method of nucleic acid measurement is appropriate, three issues are critical: specificity, sensitivity, and interfering substances. Based on these features different detection techniques are to be followed.

Traditional Detection Methods:

UV spectroscopy

Majority of bio-molecules intrinsically absorb light in the ultraviolet and not in the visible range. This property of UV absorbance can be used to quickly estimate the concentration and purity of DNA and RNA (also proteins) in a analytical sample. The amount of DNA in a sample can be estimated by looking at its absorbance at a wavelength of 260nm or 280nm (in the UV region). Purines and pyrimidines have absorbance maxima slightly below and above 260 respectively. Thus the absorbance maxima of different fragments of DNA vary somewhat depending on their subunit composition. Contaminants like proteins exhibit two absorbance peaks, one between 215-230 nm (due to peptide bonds absorption) and at about 280 nm (absorption by aromatic amino acids-tyrosine, tryptophan and phenylalanine). Remember that although proteins have little absorbance at 260 nm, both proteins and nucleic acids absorb light at 280 nm. That is the reason why, if nucleic acids and proteins are mixed in the same sample, their spectra interfere (overlap) with one another.

Table: The relationship between concentration of DNA, RNA, Protein and absorptivity are as below:

Sample	Absorbance value	Quantity (approximate)
Double-stranded DNA	1at 260 nm	50 µg/mL
Pure single-stranded DNA	1 at 260 nm	33 µg/mL
Pure RNA	1 at 260 nm	40 µg/mL
Pure protein (vary in	1 at 280 nm	1 mg/mL
general)	1.2-1.35	1 mg/mL
Antibodies

The purity of a solution of nucleic acid is determined by measuring the absorbance of the solution at two wavelengths, usually 260 nm and 280 nm, and calculating the ratio of A260/A280. Value of this ratio is 2.0, 1.8and 0.6 for pure RNA, DNA and protein respectively. A ratio of less than 1.8 signifies that the sample is contaminated with protein or phenol and the preparation is not proper.

Hyperchromicity is the phenomenon of increment of absorbance when any material is exposed to UV light.The most well known application is the hyperchromicity of DNA that occurs when the DNA duplex is denatured and melting of DNA occurs. The UV absorption is increased when the duplex DNA strands are being separated into single strands, either by heat or by addition of denaturant or by increasing the pH level. On the contrary, decrease of absorbance peak is called hypochromicity.

Ethidium Bromide Staining:

The IUPAC name for EtBr is 2, 7-diamino-10-ethyl-9-phenylphenanthridiniumbromide. It is commonly used as a fluorescent dye for nucleic acid staining. It binds as well as intercalates with nucleic acid (mainly with major and minor groove of DNA) and gives orange fluorescence under UV radiation from 500 – 590 nm. Usually EtBr may be added in warm agarose gel before solidification. When DNA or RNA samples are run in agarose gel electrophoresis EtBr molecules will bind with nucleic acids and help in detection under UV light. The post staining can also be done for nucleic acid detection.

Ethidium bromide (EtBr) is a potent mutagen and carcinogen. Dyes to stain nucleic acids such as SYBR green, SYBR Safe etc are safer to use instead of EtBr.

Polyacrylamide gel can be used for separation of Nucleic acids and post staining of the gel with EtBr is done for detection. The sensitivity of EtBr staining is of nano-molar level.

1.2.3 Silver Staining:

Silver staining based on reduction of silver nitrate is more sensitive than ethidium bromide for double stranded DNA, as well as detection of single stranded DNA or RNA with a good sensitivity (in picogram level). It is based on the reduction of silver cations to insoluble silver metal by nucleic acids. This chemical reaction is insensitive to the macrostructure of the DNA molecule. Reduced silver molecules deposit in the gel around the DNA bands, creating a dark black band like image (i.e. “latent image”). Then the latent image can be developed to visualize by soaking the gel in a solution of silver cations (Silver nitrate) and a reducing agent (eg. formaldehyde). The silver granules in the latent image catalyze the further reduction and deposition of silver from the solution. Bands manifest as dark brown or black regions which appear before significant background develops. Development is stopped by altering the pH of the gel to a point where silver reduction is no longer favored.

Fig. 3-1.3.3 Representation of a silver staining of DNA

Lane 1: DNA of lesser concentration

Lane 2: DNA of higher concentration

Lane 3: Low molecular weight ladder

1.2.4 Nanodrop:

Detection assays are persistently being developed that use progressively smaller amounts of nucleic acid, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform micro-volume quantitation. The patented NanoDrop microvolume sample retention system functions by combining fiber optic technology and natural surface tension properties to capture and retain small amounts of sample . This is a novel technology which allows us to measure nano-liter volumes (pico concentration) of the nucleic acid (DNA or RNA) sample. It is a type of spectrophotometer with a smaller sample size (as much less as 1-2 microlitre) requirement and higher sensitivity (even upto pico molar level). This is also a time saving technology widely used in basic molecular biology research.

Fig. 3-1.3.4 NanoDrop® ND-1000 Spectrophotometer

(Source: http://www.biotech.wisc.edu/facilities/gec/equipment/nanodropnd1000)

1.2.5 Fluorometric Quantification:

Fluorometric method applies fluorescence dyes to detect the presence and concentration of a class of nucleic acid (DNA or RNA). This method is more sensitive and less prone to contaminants than UV spectroscopy.

An assay using Hoechst 33258 dye is specific for DNA because it is less sensitive to detect RNA. This assay is commonly used for rapid measurement of low quantities of DNA, with a detection limit of ~1 ng DNA. It is useful for the measurement of both small and large amounts of DNA (verifying DNA concentrations prior to performing electrophoretic separations and Southern blots) because this assay accurately quantifies a broad range of DNA concentrations from10 ng/ml to15 μg/ml. The Hoechst 33258 assay can also be employed for measuring products of the polymerase chain reaction (PCR) synthesis.

Hoechst 33258 is non-intercalating reagent and binds to the minor groove of the DNA with a preference for AT sequences (Portugal and Waring, 1988). The binding to the minor groove has is dependent upon a combination of structural preferences (eg., the minor groove with a series of contiguous AT base pairs is more narrow).(Neidle (2001) ,Like other minor groove binding ligands, Hoechst 33258 is positively charged and thus form electrostatic interaction with the negative potential of stretch of AT base pairs. Upon binding to the minor groove of the double helix DNA, the fluorescence characteristics of Hoechst 33258 change dramatically, showing a large increase in emission at ~458 nm.

According to Daxhelet et al, the fluorochrome 4’,6-diamidino-2-phenylindole (DAPI) has similar characteristics to H33258 and binds to the minor groove as well. DAPI is also appropriate for DNA or RNA quantitation, although it is not as commonly used as Hoechst 33258. DAPI is excited with a peak at 344 nm. Emission is detected at around 466 nm for DNA, similar to Hoechst 33258 but for RNA the peak shifts to ~500 nm.

1.2.6 Hybridization-Based Techniques

Hybridization-Based Techniques for nucleic acid detection has higher resolution (down to the actual nucleotide sequence) and utilizes "probe sequence" for DNA or RNA, and when it finds its intended target, binds to it by hybridization process. Since the "probe" is attached to a label such as a fluorescent chemical isotope, the bound sequence can thus be visualized. This is the principle of Southern Blotting method, as well as its variants, and requires the target nucleic acid sequence to undergo separation by agarose-electrophoresis, transferred to an appropriate membrane (typically nitrocellulose), and then treated with a solution containing the labeled probe (fluorescent or colorimetric).

Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. Since the probe specifically binds to its target, the membrane can be documented and analyzed so that the bound target sequences can be identified and studied. The detailed methodology is described in later chapter (Module-4Lecture 3).

FISH (Fluoroscence in-situ hybridization) for visualization of nucleic acids developed as an alternative to older methods that used radio labeled probes (Gall and Pardue, 1969). Several drawbacks of isotopic, radiolabeled hybridization stimulated the development of novel techniques like FISH. In the year 1980, RNA was first directly labeled on the 3’ end with fluorophore was used as a probe for specific DNA sequences (Bauman et al., 1980). Enzymatic incorporation of fluorophore-modified bases throughout the probe has been widely used for the preparation of fluorescent probes. The use of amino-allyl modified bases (Langer et al., 1981), which could later be conjugated to any sort of hapten or fluorophores, was critical for the development of in situ technologies because it allowed production of an array of low-noise probes by simple chemistry. Methods of indirect detection result into higher magnitude signal output by the use of secondary reporter molecules that bind to the hybridization probes.

In the early 1980s, assays like nick-translated, biotinylated probes, and secondary detection

by streptavidin conjugates were used for detection of DNA (Manuelidis et al., 1982) and mRNA (Singer and Ward, 1982) targets. Later, advanced labeling of synthetic, single-stranded DNA probes allowed the chemical preparation of hybridization probes carrying enough fluorescent molecules to allow direct detection (Kislauskis et al., 1993). At this current era, based on these reaction themes of indirect and direct labeling have since been introduced, a wide spectrum of nucleic acid detection schemes are available.

Whereas the initial development of FISH involved expansion of the types of probe and number of detectable targets, the outlook for future development of fluorescence techniques will include expansion of the subjects of investigation like in clinical, diagnostics, forensic applications.

Other detection methods include amplification of target region by polymerase chain reaction and various forms of chemiluminescent, fluorescent or radioactive detection method. Specific technical or sample requirements are present for each method, and the original purpose for detection the nucleic acid will determine which of the methods is most suitable.

Bibliography:

Bauman, J. G., Wiegant, J., Borst, P. and van Duijn, P. (1980). A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA. Exp. Cell Res. 128, 485-490.

Daxhelet, G.A., Coene, M.M., Hoet, P.P., and Cocito, C.G. 1989. Spectrofluorometry of dyes with DNAs of different base composition and conformation. Anal. Biochem. 179:401-403.

Fluorometric Quantitation of DNA Using Hoechst 33258 Cold Spring HarbProtoc; 2006; doi:10.1101/pdb.prot4458.

Gall, J. G. and Pardue, M. L. (1969). Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA 63, 378-383.

Kislauskis, E. H., Li, Z., Singer, R. H. and Taneja, K. L. (1993). Isoform specific 3’untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell Biol. 123, 165-172.

Langer, P. R., Waldrop, A. A. and Ward, D. C. (1981). Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637.

Manuelidis, L., Langer-Safer, P. R. and Ward, D. C. (1982). High-resolution mapping of satellite DNA using biotin-labeled DNA probes. J. Cell Biol. 95, 619-625.

Molecular Cloning- A laboratory manual-David W. Russell & Joseph Sambrook.

Neidle, S. DNA minor-groove recognition by small molecules. 2001. Nat. Prod. Rep. 18:291-309.

Portugal, J. and Waring, M.J. (1988). Assignment of DNA binding sites for 4,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258): A comparative footprinting study. Biochem. Biophys. Acta 949:158-168.