Recently, controlled-pore glass columns that introduces a dabcyl moiety at the 3' end of an oligonucleotide have become available, which enable the synthesis of molecular beacons completely on a DNA synthesizer. Alternatively, the starting material for the synthesis of molecular beacons is an oligonucleotide that contains a sulfhydryl group at its 5' end and a primary amino group at its 3' end. Dabcyl is coupled to the primary amino group utilizing an amine-reactive derivative of dabcyl. The oligonucleotides that are coupled to dabcyl are then purified. The protective trityl moiety is then removed from the 5'-sulfhydryl group and a fluorophore is introduced in its place, using an iodoacetamide derivative.
High-pressure liquid chromatograph
Spectrofluorometric thermal cycler with a capacity to monitor fluorescence in real time
0.1 M sodium bicarbonate, pH 8.5
0.2 M sodium bicarbonate, pH 9.0
HPLC Buffer A: 0.1 M triethylammonium acetate, pH 6.5, filtered and degassed
HPLC Buffer B: 0.1 M triethylammonium acetate in 75% acetonitrile, pH 6.5, filtered and degassed
TE buffer: 1 mM EDTA, 10 mM Tris-HCl, pH 8.0
Molecular beacon buffer: 1 mM MgCl2, 20 mM Tris-HCl, pH 8.0
Figure 1. Chromatographic separation of oligonucleotides coupled to dabcyl. The blue line represents absorption at 260 nm and the red line represents absorption at 491 nm. The oligonucleotides in peaks A and B do not contain trityl moieties, whereas the oligonucleotides in peaks C and D are protected by trityl moieties. The oligonucleotides in peaks B and D are coupled to dabcyl, whereas the oligonucleotides in peaks A and C are not coupled to dabcyl. Peak D should be collected.
Figure 2. Chromatographic separation of oligonucleotides coupled to both dabcyl and fluorescein. The blue line represents absorption at 260 nm and the red line represents absorption at 491 nm. The oligonucleotides present in peak E are not coupled to fluorescein, whereas the oligonucleotides in peak F are coupled to fluorescein. Peak F should be collected.
An example of a hybridization reaction performed for the determination of the signal-to-background ratio is shown in Figure 3. The signal-to-background ratio in this example was 190. Usually the ratio ranges from 30 to 200. An example of a thermal denaturation profile is shown in Figure 4. The probe-target hybrid denatures at 58 °C and the stem of the molecular beacon denatures at 64 °C. In the range of 10 °C to 50 °C, the free probe has very little fluorescence, whereas the target-bound form is fluorescent. The sequence of the molecular beacon used throughout this section is: fluorescein-5'- GCG AGC TAG GAA ACA CCA AAG ATG ATA TTT GCT CGC -3'-dabcyl, where underlines identify the arm sequences.
Figure 3. The spontaneous fluorogenic response of molecular beacons to the addition of target. The first segment of the data is due to the fluorescence of the buffer, the second segment is due to the fluorescence of the buffer containing the molecular beacons, and the third segment shows the increase in fluorescence that occurs upon the addition of target oligonucleotides.
Figure 4. Thermal denaturation profiles of a molecular beacon (red-dotted line) and the hybrid formed between the molecular beacons and its oligonucleotide target (blue-dashed line). The profiles indicate that this molecular beacon can be used below 55 °C.
Utilize molecular beacons that are complementary to a sequence in the middle of the expected amplicon. The length of their arm sequences should be chosen so that a stem is formed at the annealing temperature of the polymerase chain reaction. The length of the loop sequence should be chosen so that the probe-target hybrid is stable at the annealing temperature. Whether a molecular beacon actually exhibits these design features is determined by obtaining thermal denaturation profiles, as detailed in the previous section. Molecular beacons with appropriate thermal denaturation characteristics are included in each reaction at a concentration similar to the concentration of the primers. During the denaturation step, the molecular beacons assume a random-coil configuration and fluoresce. As the temperature is lowered to allow annealing of the primers, stem hybrids form rapidly, preventing fluorescence. However, at the annealing temperature, molecular beacons also bind to the amplicons, undergo conformational reaorganization, and generate fluorescence. When the temperature is raised to allow primer extension, the molecular beacons dissociate from their targets and do not interfere with polymerization. A new hybridization takes place in the annealing step of every cycle, and the intensity of the resulting fluorescence indicates the amount of accumulated amplicon. In the procedure below, the synthesis of an 84-nucleotide-long amplicon is monitored with the same molecular beacon whose synthesis and characterization was described in previous section.
Figure 5 shows the level of fluorescence as a function of the number of temperature cycles completed.
The level of fluorescence is proportional to the amount of amplicons present in each cycle. The
reaction that did not contain any template, did not show any rise in fluorescence. The number of temperature
cycles required before the fluorescence signal becomes detectable over the background is inversely
proportional to the logarithm of the initial number of template molecules. This
relationship is true over a wide range of template concentrations.
Figure 5. (A) The threshold cycle (Ct) is the cycle at which the fluorescence rises significantly above the background. The fluorescence increases as the molecular beacons bind to the amplification products that accumulate during each successive cycle. In the early cycles of amplification, the change in fluorescence is usually undetectable, but at some point during amplification, the accumulation of amplified DNA results in a detectable change in the fluorescence of the reaction mixture. The threshold cycle number decreases as the number of target molecules initially present in a reaction increases. (B) The standard curve can be used to determine the starting amount of an unknown template, based on its threshold cycle. Given known starting amounts of the target, a standard curve can be constructed by plotting the log of the starting amount versus the threshold cycle. The threshold cycle is inversely proportional to the logarithm of the number of target molecules initially present.
Detailed descriptions on the design, synthesis and application of molecular beacons appeared in:
Vargas DY, Marras SAE, Tyagi S, and Kramer FR. (2018) Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays That Utilize SuperSelective Primers for the Detection of Rare Somatic Mutations. The Journal of Molecular Diagnostics, 20, 415-427
Schlachter S, Chan K, Marras SAE, and Parveen N (2017) Detection and differentiation of lyme spirochetes and other tick-borne pathogens from blood using real-time PCR with molecular beacons. Methods in Molecular Biology 1616: 155-170.
Catrina IE, Bayer LV, Yanez G, McLaughlin JM, Malaczek K, Bagaeva E, Marras SAE, and Bratu DP (2016) The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol 13: 1117-1132.
Vargas DY, Kramer FR, Tyagi S, and Marras SAE. (2016) Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS ONE 11, e0156546.
We describe the use of SuperSelective primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time multiplex PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies), thereby providing information that can be used to modify therapy for individual patients, prolonging (and improving the quality of) life.
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