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Custom DNA Synthesis - Technical Corner Quantifying DNA Through its aromatic purine and pyrimidine heterocycles-containing moiety (fig. 6), oligos absorb UV light thus allowing an easy mean of quantitation in solution. Click here to enlarge Fig. 6.

First approximation, for all practical purposes
As a first approximation, satisfactory in most Biological applications, Optical Density readings at 260 nm (OD260: absorbance of 1-ml solution in a 1-cm path cuvette) are converted to mass as shown below. This mass to absorbance ratio value is derived from an averaged Molar Extinction Coefficient value of 10.000 L/mol/cm for DNA in aqueous solutions.

Molecular weight determination
M.W. - 1st approximation*: MW = 330 x (# bases)
(* DNA-oligos with all-phosphodiester linkages in its acidic forms)

M.W. - 2nd approximation*:
MW= [328.2 x (#G)] + [312.2 x (#A)] + [303.2 x (#T)] + [288.2 x (#G)] - 61.0
(* DNA-oligos with all-phosphodiester linkages in its acidic form)

A more accurate quantitation of oligos from absorbance readings at 260 nm depends upon the determination of the molar extinction coefficient at 260 nm (e260). The latter is dependent upon sequence and pairwise assembly of the sequence and a number of software are available for extinction coefficient determination.
OD260 = e260 x (mmoles of oligo) (Path=1cm, Volume=1ml)
Note: For MW= 330 & e260= 10 000, one gets the all purpose value of 33 µg / OD260

In some instances of strong base composition bias (e.g. homopolymers), one may use a more accurate weight to absorbance ratio.

References
• Basic Principle in Nucleic Acids Chemistry, Academic Pres (1978)
• Fasman, G.D., ed., Handbook of Chemistry and Molecular Biology, Nucleic Acids - Vol. I, CRC Press (1978)

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Degenerate primers

Degenerate or mixed probe are often required when protein amino acid sequence is the only information available about the gene / gene product under investigation. Due to the degeneracy of the genetic code, several distinct DNA sequences can code for one given amino acid sequence. Hence, a set of probes differing by several bases at several positions must be prepared, with only one of which will perfectly match the targeted protein coding DNA sequence. The latter single probe can be largely under-represented.

I.U.B. codes for degenerate positions:

R=	A/g
M=	A/C
W=	A/T
Y=	C/T
S=	g/C
K=	g/T
H=	A/T/C
D=	g/A/T
B=	g/T/C
V=	g/A/C
N=	g/A/T/C

General considerations in probe design
• In all cases, stringent hybridization conditions should be used for optimum discrimination.
• One should avoid sequences containing aa specified by more then two codons. If large pools of primers cannot be avoided or you must use an amino acid with six possible codons, synthesize two pools of oligos.
• One should use a universal base (e.g. deoxyinosine) for some or all positions with the 4-level degeneracy (i.e. N).
• One could consider performing a double hybridization experiment within two independent region even close to each other in order to minimize false positives.

Deoxyinosine
In view of the complexity and heterogeneous nature of probe sequences generated when oligos with ambiguous positions are prepared, alternative "universal" base-pairing molecules have been developed for synthetic incorporation into oligos. The use of "universal" or "neutral" bases ensures that every probe present within the hybridization mix will be able to hydrogen bond to the target sequence. However, the drawback of universal bases is as intrinsic as their advantages, they can hydrogen bond with any base. It is therefore advised to use universal bases in place of a 4-base degeneracy (i.e. N) position only.

2'-Deoxyinosine is currently the most widely used "universal" base nucleoside (Fig. 7).
Hypoxanthine (the base moiety of the inosine), is a naturally occurring base, widely found as the first base in the anticodon loop of tRNAs. In the vast majority of tRNA analyzed, none have anticodon starting with 5'-A. In fact, whenever A appears at this position in the RNA transcript, it is further processed through deamination to hypoxanthine.

Click here to enlarge Fig. 7.

Reporter-Quencher Probes
Molecular Beacons (1) are newly developed tools for fast, sensitive, non-radioactive, and quantifyable DNA hybridization in homogeneous solutions.
Molecular Beacons are oligonucleotides consisting of self-complementary ends flanking a loop containing the sequence of interest (Fig 8). The 5' and 3' ends are tagged with a fluorophore and a suitable quencher, respectively. In the absence of target DNA, the Molecular Beacon oligo forms a stem-loop structure bringing in close sterical proximity the fluorescent dye and the quencher moieties resulting in a non-fluorescent solution. However, upon adding the target DNA complementary to the loop part of the Molecular Beacon oligo, one sees instantaneous appearance of fluorescence emission under suitable experimental conditions resulting from the hybridization of the Molecular Beacon probe to its target and the spreading apart of the quencher and fluorophore moieties. Molecular Beacons have been successfully utilized in mRNA quantitation, real-time monitoring et quantitation of PCR amplified products, rRNA-based microbial discriminative probing (1,4,5,6).

Click here to enlarge Fig. 8.

Reporter - Quencher: Principle
There are two main processes that leads to quenching (reduction in fluorescence quantum yields): collisions with local energy exchange and long-range radiation-based called Energy Transfer on which Molecular Beacons principle is based.
As originally confirmed using tagged poly-prolines of various length (2), when in close molecular proximity (typically 10-100 Å), the emitted light (energy) of the excited fluorophore is absorbed by a quencher of overlapping absorption spectrum.
The efficiency of excited-state energy transfer (i.e.. Quenching) is a function of molecular distance separating the Fluorophore-Quencher pair as well as spectral properties of the pair including extent of spectral overlap between fluorescence emission and absorption.

Dabcyl-Fluorescein: pair of choice for Molecular Beacons.
The non-fluorescent dabcyl chromophore has proven very useful as a quencher because of its broad visible absorption spectra of its bioconjugates. Fluorescein exhibits good overlapping emission spectra with dabcyl and gives a strong signal. Dabcyl-Fluorescein appears to be a good Reporter-Quencher pair for Molecular Beacons probes with an excitation at 490 nm and fluorescence reading at 520nm. Note that depending upon the experimental conditions, one may need to optimize the emission wavelength (from 515 up to 535nm).

Practical considerations
-Specificity. Molecular Beacons are highly discriminatory. 80% decrease in Fluorescence intensity was observed with an oligonucleotide containing a single mismatch to the loop region of a 5 molar excess of Molecular Beacon compared to the complementary sequence (6).
-Probe Design - The stem portion. A five-base-pair stem containing 1 A/T appears to be optimum in the presence of at least 1 mM MgCl2 for stabilization of the stem-loop structure. We have good experience with the following sequence (Fig. 9):

5'- gCAgC—LOOP—gCTgc-3'

Note: An all GC stem sequence appears to induce an atypical behavior and should be avoided.

Click here to enlarge Fig. 9.

-Probe Design - The loop portion. Any length typically from 15 to 25 nt can be used, depending upon the target. A GC content over 40% would allow you to design a loop of 15 nt. We have found that a 20% GC content in the target sequence of 20% would require a loop of 25 nt.
-Experimental. Hybridization is usually performed in 100 mM Tris, pH=7.5 and 1 mM MgCl2. MgCl2 is crucial for stabilization of the stem structure. Final concentration of Molecular Beacons should be within 50-200 nM depending upon the assay. For real-time PCR product detection, 150-200 nM (10 to 50 pmol) of probe is often optimum. Simpler mixtures would require 50nM (2-5 pmol). For other applications than PCR, one should heat the sample containing the target and the probe for 1-5 min at 100ūC and cool it down to RT over time before taking fluorescence measurements.

References
(1) Tyagi, S. et al. (1996), Nature Biotech., 14, 303
(2) Stryer, L. (1978), Ann. Rev. Biochem., 47, 819
(3) Van der Meer, B.W. et al. (1994), Resonnance Energy Transfer Theory and Data, VCH Publishers
(4) Blok,HJ. et al. (1997), Mol. Cell. Probes, 11, 187.
(5) Gao, W. et al. (1997), Mol Microbiol, 25, 707
(6) Schofield et al. (1997), App. Env. Microbiol., 63, 1143