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.
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.
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
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
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