Sloppy molecular beacon probes.
We designed and synthesized four different sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore (Fig. ). The four fluorophores (fluorescein on probe A, Alexa Fluor 546 on probe B, Alexa Fluor 594 on probe C, and Cy5 on probe D) were chosen because their fluorescence signals are readily distinguishable by the spectrofluorometric thermal cycler in which the PCR assays were carried out. Although each of the four probes was designed to hybridize to the target sequences in the amplicons of all of the 27 mycobacterial species, none of the four probes was perfectly complementary to any of the target sequences. Instead, the probe sequence in each of the four molecular beacons was selected so that its complementarity with the 27 different target sequences varied from species to species.
FIG. 1. Schematic representation of the four sloppy molecular beacon probes. Each probe possessed a different sequence and a differently colored fluorophore, enabling the four probes to be used simultaneously in the same reaction mixture and to be distinguished (more ...)
Ideally, the probe sequence of each sloppy molecular beacon should be chosen so that its degree of complementarity with the target sequence from each species is different. However, the number of mismatched base pairs should not be so great that the hybrid cannot form. For the experiments reported here, we selected the sequences of probes A and B to be similar to each other, differing by only three nucleotides and forming hybrids that contained between 1 and 11 mismatched base pairs depending on which mycobacterial target sequence was present in the amplicon. On the other hand, we selected the sequences of probes C and D to differ from each other by 11 nucleotides and form hybrids that contained between 1 and 15 mismatched base pairs. The positions of the nucleotides at which the sequences of the four probes differed from one another were chosen to correspond to the nucleotides in the set of 27 target sequences that showed the most variation in the hope that each probe would respond differently to each target.
Preliminary hybridization experiments.
We prepared 27 hybridization reaction mixtures, each containing an excess of 1 of the 27 different target oligonucleotides; we also prepared a control reaction mixture that contained no targets. Each reaction mixture also contained the four differently colored molecular beacons, the concentration of each being chosen so that they would all produce fluorescence signals of approximately the same intensity. Hybrids were formed by being incubated at 25°C in the same buffer that we use for PCR assays. The Tm values of the four different hybrids formed in each reaction were determined automatically by a Bio-Rad iQ5 spectrofluorometric thermal cycler, which slowly increased the temperature from 25°C to 95°C in 1°C steps.
Figure shows the probe-target hybrid denaturation profiles that were obtained for the hybrids formed by probe A with oligonucleotide target sequences from seven different mycobacteria. At low temperatures, a strong fluorescence signal was produced in each tube, indicating that the molecular beacon probe was hybridized to all seven species-specific targets. However, as the temperature was slowly increased, the intensity of the fluorescence signal produced by each species eventually dropped, indicating that the molecular beacon probe had dissociated from its target and formed a nonfluorescent conformation. The key observation here is that the temperature at which each species-specific probe-target hybrid dissociates is different, depending on the number of mismatched base pairs and on the location of those mismatched base pairs in the hybrid.
It is common to think of fluorescent hybridization probes as tools to determine whether a particular target sequence is present or absent in a sample by observing whether a fluorescence signal occurs or does not occur after incubation of the sample with the probe. However, these results demonstrate that sloppy molecular beacon probes form a fluorescent hybrid with many different target sequences even though they are not perfectly complementary to the targets.
The beauty of sloppy molecular beacons is that the stability of the probe-target hybrids that they form provides a characteristic Tm value that depends on the identity of the target.
Utilizing the data shown in Fig. , the derivative of fluorescence intensity with respect to temperature was plotted as a function of temperature, with negative values plotted above the x axis (Fig. ). In this plot, the decrease in fluorescence intensity that occurs when a hybrid dissociates is seen as a peak that rises and then falls, with the highest rate of dissociation (the top of the peak) occurring at the hybrid's Tm. In order to easily compare the results obtained with different hybrids, the data that occur within approximately 2°C of each peak in Fig. were normalized so that the values of the derivatives were plotted between 0 and 1, as shown in Fig. .
The results of these preliminary hybridization experiments are graphically summarized in the four panels of Fig. . For each sloppy molecular beacon probe, the stability (Tm) of the hybrid that it forms with each of the 27 species-specific oligonucleotides is plotted as a function of the number of mismatched base pairs that occur in that hybrid. The results highlight the dependence of Tm on the relatedness of the nucleotide sequence of the probe to the nucleotide sequence of the target. In general, the more mismatched base pairs that occur in a hybrid, the lower its Tm value. These results demonstrate that the Tm obtained from the hybrid formed by any one of the probes provides information that helps to identify the target species to which the probe is hybridized but that Tm, in and of itself, is not sufficient to uniquely identify the target. However, the combination of the four Tm values obtained for the same target with four different probes provides much more information about the identity of the target (see, for example, the species-specific results highlighted by the colored dots in Fig. ). These preliminary results illustrate the underlying principle of obtaining a species-specific signature through the use of a series of independent measurements. For example, if each of the sloppy molecular beacon probes was capable of providing only 10 distinguishable Tm values, then the results from four different probes, taken together, would provide 10,000 different species-specific signatures (10 × 10 × 10 × 10).
Use of LATE-PCR.
The simultaneous use of sloppy molecular beacon probes in PCR assays generates hybrids that are sometimes relatively weak because they possess many mismatched base pairs. This raises a number of concerns for the selection of an effective design for the PCR assays, all of which could cause a decrease in the intensity of the fluorescence from weaker hybrids, including the following: (i) competition between probes that form strong hybrids and probes that form weak hybrids might diminish the number of weaker hybrids that form; (ii) competition between the probes and the complementary amplicon strands for binding to the target strands might diminish the number of weaker hybrids that form; and (iii) secondary and tertiary structures that are present in the target strands might diminish the formation of weaker hybrids. We therefore decided to utilize linear-after-the-exponential (LATE)-PCR, which is an efficient asymmetric PCR format (13
), in which so many target strands are synthesized that their number exceeds the number of sloppy molecular beacon probes present in the assay and in which many more target strands are synthesized than complementary strands. By eliminating sources of competition for the binding of probes to target strands, weaker hybrids are more abundant, and their fluorescence is therefore more likely to be sufficiently intense for their Tm
to be measured.
Determination of species-specific signatures.
We carried out 27 LATE-PCR assays, each containing the four sloppy molecular beacon probes and genomic DNA from 1 of the 27 different mycobacterial species, and we carried out control assays that did not contain any mycobacterial DNA. After the completion of amplification, the Tm values of the hybrids formed by the four differently colored sloppy molecular beacon probes with each species-specific target amplicon were determined automatically by the Bio-Rad iQ5 spectrofluorometric thermal cycler.
To illustrate the nature of the results that were obtained, the denaturation profiles of the hybrids formed by probe A with seven different mycobacterial target amplicons are shown in Fig. . As can be seen, the lower the stability of the hybrid (as evidenced by locations where a drop in intensity occurred), the weaker the overall intensity of the fluorescence signal. The lower fluorescence intensity of the less-stable hybrids was not due to competition among the four probes, since the same results were obtained in preliminary experiments in which only probe A was present. Moreover, the reduction in fluorescence intensity seen in weaker hybrids formed from amplicon strands was not seen in corresponding hybrids formed from oligonucleotides, implying that the lower fluorescence of the less-stable probe-amplicon hybrids is due to the presence of secondary and tertiary structures in the amplicon strands that restrict the access of the probes to the target sequence. Despite the lowering of the fluorescence intensity from the less-stable hybrids, the results demonstrate that the fluorescence signal from all seven hybrids was sufficiently intense to enable each hybrid's characteristic Tm value to be determined.
See Tables SA, SB, SC, and SD in the supplemental material for a comprehensive listing of the results that were obtained with amplicons. In the supplemental material, there is a different table for each of the four sloppy molecular beacon probes, and each table shows the nucleotide sequence of each species-specific target and highlights in black letters those nucleotides that are not complementary to the corresponding nucleotide in the probe sequence. The Tm value obtained for each of the hybrids is listed in the right-hand column, and the results are shown in the order of hybrid stability, with the most stable hybrids listed at the top of each table. These results show that, in addition to there being a roughly inverse correlation between the number of mismatched base pairs in a hybrid and its stability, Tm values are affected by the identity of the mismatches, the identity of neighboring base pairs, the location of the mismatches within the probe-target hybrid, and whether or not the mismatches occur in a run of adjacent mismatches. The results also show that the fluorescence intensity of probe-target hybrids that possess more than 10 mismatched base pairs was too low to obtain useful Tm values. Despite this observation, the set of three or four Tm values that was obtained for each species is sufficiently unique to constitute a species-specific signature that identifies the species that was present in each sample. Table summarizes the results obtained for each of the 27 mycobacterial species that were tested.
Figure shows, for each of the 27 mycobacterial species, the normalized derivatives obtained by melting the hybrids formed by the PCR amplicons with each of the differently colored sloppy molecular beacon probes. An examination of these results shows that the genomic DNA from each species produces a combination of Tm values that distinguishes that species from all of the other species tested. Even mycobacterial species whose 39-nucleotide-long target sequences are extraordinarily similar produce reproducibly distinguishable species-specific signatures.
For example, the target sequences from M. asiaticum and M. tuberculosis differ from each other only by the substitution of a single adenosine for a guanosine, yet the Tm values for the hybrids formed by M. tuberculosis amplicons with probes B and C are consistently 1°C higher than the Tm values for the corresponding hybrids formed by M. asiaticum, and the Tm values for the hybrids formed by M. tuberculosis amplicons with probes A and D are consistently 2°C higher than the Tm values for the corresponding hybrids formed by M. asiaticum. In order to illustrate the precision of the Tm measurements, every PCR assay was repeated three times, and all three multiprobe fluorescence signatures obtained for each mycobacterial species were virtually superimposable.
Effect of initial mycobacterial DNA concentration on the species-specific signature.
A particularly attractive aspect of exponential gene amplification techniques, such as PCR, is that over a wide range of amounts of template DNA initially present, the final amounts of amplified DNA do not vary very much. This is a very useful attribute when the amplicons are used to form probe-target hybrids for the measurement of hybrid stability, since the Tm of the hybrid is affected by target concentration. To illustrate this desirable feature, we carried out five different LATE-PCR assays, each initiated with a different number of M. chubuense genomic DNA molecules. The reactions were initiated with as little as 100 molecules of genomic DNA and as many as 1,000,000 molecules of genomic DNA. After amplification, the denaturation profiles of the four differently colored probe-target hybrids that were present in each reaction mixture were determined.
The results (Fig. ) demonstrate that the number of thermal cycles required to complete the symmetric phase of each LATE-PCR was, as expected, an inverse linear function of the logarithm of the number of molecules of genomic DNA initially present. Significantly, the normalized derivatives obtained from the five PCRs were virtually superimposable, despite the wide range of initial DNA concentrations tested (Fig. ), confirming that the Tm values of the hybrids formed by sloppy molecular beacon probes with the amplified DNA of a bacterial species yield a characteristic species-specific signature irrespective of the initial DNA concentration.
Test of blinded mycobacterial DNA samples.
To see whether species-specific signatures can be used to reliably identify mycobacterial species, we utilized the four sloppy molecular beacon probes in LATE-PCR assays to test 55 blinded DNA samples that were prepared from mycobacteria isolated from patients over a 2-year period at the Memorial Sloan-Kettering Cancer Center. The set of Tm values obtained from each of the 55 samples was compared to the 27 previously determined species-specific signatures listed in Table . The signatures obtained from 53 of the 55 samples correctly matched the signature of 1 of the 27 species already in our database. However, the signatures obtained from two of the samples did not match any of the 27 previously determined signatures. Sequence analysis of the amplicons present in these two discrepant samples indicated that one sample (probe A Tm, 65°C; probe B Tm, 58°C; and probe D Tm, 55°C) was Mycobacterium gordonae, which was not in our database, and the other sample (probe A Tm, 61°C; probe B Tm, 59°C; probe C Tm, 54°C; and probe D Tm, 54°C) was a variant of M. xenopi that possessed a single-nucleotide substitution in its target sequence, also not in our database. The two new species-specific signatures can now be added to our database, illustrating how the scope of these assays can be enhanced as additional species are tested. These results are summarized in Table .