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Abstracts of Oak Ridge Posters |
1 PerkinElmer Life and Analytical Sciences, Wallac, PO Box 10, FIN-20101 Turku, Finland
aauthor for correspondence: fax 358-2-2678-357, e-mail alice.ylikoski{at}perkinelmer.com
Laboratories performing genetic screening studies need simplified methods that allow automation for nucleic acid analysis. Ideally, a method should have as few handling steps as possible and allow the use of a closed-tube assay format to reduce the risk of PCR contamination. Different homogeneous assay chemistries have been exploited to integrate amplification and detection to allow simple nucleic acid analysis with minimal post-PCR handling (1)(2)(3)(4)(5).
Double-stranded DNA probes consisting of a 5'-terminus-labeled fluorescent strand and a complementary strand labeled on the 3' terminus with a quencher have been exploited in homogeneous hybridization (2)(6)(7). Fluorescence signal is generated when the fluorophore strand hybridizes with the target. When the quencher strand hybridizes with the fluorophore strand, the signal from the fluorophore is quenched. Morrison et al. (6) used probes consisting of strands of equal length and a competitive hybridization technology, whereas others have exploited probes consisting of strands with different lengths for detection of PCR products (2)(7).
The use of stable and fluorescent lanthanide chelates has enabled the development of homogeneous assay technologies based on time resolution (8). Time-resolved fluorescence-quenching assays (TR-FQAs), based on energy transfer from a lanthanide chelate to a nonfluorescent quencher, have been applied in various assays of hydrolyzing enzymes (9)(10) and are commercialized as TruPointTM. The first homogeneous PCR assays with lanthanide-labeled probes used an environmentally sensitive terbium chelate, the fluorescence intensity of which changes on probe hybridization (3)(11). Subsequently, the technology was further improved by use of additional quencher probes (12). Recently, a combination of quenching and competitive hybridization has been applied for end-point detection of PCR products (13).
The aim of the present study was to compare two homogeneous PCR assay approaches based on a dual-label TR-FQA: one in which the probes hybridize during PCR cycling (called TruPoint-PCR) and the other in which the probes hybridize with the single-stranded target produced by asymmetric PCR only after PCR cycling (called competitive TruPoint-PCR). Labeling of the oligonucleotides during solid-phase synthesis allowed simple and straightforward production of the probes. The performance of the assay was demonstrated with known genomic DNA samples, and its applicability was demonstrated in end-point and real-time monitoring of target amplification from as little as 1 ng of genomic DNA.
Previously genotyped (14) genomic DNA samples with wild-type, carrier, and mutant status for 
F508 of the cystic fibrosis transmembrane conductance regulator gene were used. The DNA was quantified by spectrophotometric analysis at 260 nm, and the samples were stored in aliquots at –20 °C.
Oligonucleotides (Table 1
) were synthesized by use of the Expedite Nucleic Acid Synthesis System (Perseptive Biosystems). To europium probes a ligand based on the 2,2',2'',2'''-[(-2,2':6',2''-terpyridine-6,6'-diyl)bis(methylenenitrilo)]tetrakis(acetic acid) (15)(16)(17)(18) and to terbium probes a ligand based on the 2,2',2'',2'''-{[6,6'-(pyrazole-1'',3''-diyl)bis(pyridine)-2,2'-diyl]bis-(methylenenitrilo)}tetrakis(acetic acid) (16)(19) was coupled to the 5' end during oligonucleotide synthesis. The ligands were from PerkinElmer Life and Analytical Sciences, Wallac. Treatment of the oligomer with lanthanide citrate converted the oligonucleotide conjugate to the corresponding lanthanide chelate (15). A synthesis support was used to introduce a black hole quencher 1 (Biosearch Technologies) to the 3' end of the quencher oligonucleotide according to the manufacturer’s instructions.
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For TruPoint-PCR assay (Fig. 1A
), the detection probes (Table 1
) were designed to hybridize with the specific target in temperatures above the primer annealing temperature in PCR. Thus, in PCR conditions, the detection probes can hybridize with the denatured, single-stranded target before the complementary strand is elongated from the primers, which resembles the TaqMan® (Roche) assay. The quencher probe was designed to quench the signal of the remaining europium- and terbium-labeled probes on hybridization at 30 °C.
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For competitive TruPoint-PCR (Fig. 1B
), the probes were designed so that the detection-probe–target hybrids form only after PCR (at 40 °C), after which the detection-probe–quencher-probe hybrids form at room temperature. Thus, the probes do not hybridize during PCR cycling, and the difference in melting temperatures of the detection and quencher probes allows control of the competitive hybridization by temperature. In addition, asymmetric PCR was used to generate single-stranded PCR products.
TruPoint-PCR was performed in two replicates and in a total reaction volume of 50 µL containing the detection and quencher probes, primers (Table 1
), 1x Dynazyme Buffer (Finnzymes), 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates (Amersham Biosciences), and 1 U of Dynazyme II Recombinant DNA polymerase (Finnzymes) on black Thermo-Fast 96 SemiSkirted plates (ABgene) sealed with Ultra Clear Cap Strips (ABgene).
In TruPoint-PCR, thermal cycling (DNA Engine PTC-200; MJ Research) was carried out as follows: (a) for end-point PCR, 35 cycles of 95 °C for 30 s, 56 °C for 1 min, and 69 °C for 1 min, followed by 30 °C for 1 min, after which the fluorescence signals were measured through the caps at 30 °C by a Victor2 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences, Wallac); (b) for real-time PCR, 35 cycles of 95 °C for 30 s, 56 °C for 1 min, 69 °C for 1 min, and 30 °C for 10 s. Fluorescence measurement was performed at the end of the cycle as described above. Competitive TruPoint-PCR amplification differed from the TruPoint-PCR amplification with regard to probe and primer concentrations (Table 1
), PCR program, and measurement temperature. Cycling consisted of 35 cycles of 95 °C for 30 s, 56 °C for 1 min, and 69 °C for 1 min, followed by 95 °C for 8 min, 40 °C for 20 min, and 22 °C for 15 min. Europium and terbium signals were measured at room temperature.
The functionality of the probes was studied in hybridization using different concentrations of synthetic targets (Table 1
) in PCR buffer lacking primers, deoxynucleotide triphosphates, and enzyme. Hybridization with TruPoint-PCR probes was performed at 69 °C for 20 min and quenching at 30 °C for 15 min. Hybridization with competitive TruPoint-PCR probes was performed at 40 °C for 20 min and quenching at 22 °C for 15 min. After quenching, the fluorescence was measured.
The functionality of the probes was demonstrated in hybridization. Black hole quencher 1 was found to be an optimal quencher of both europium and terbium fluorescence, with 99% quenching efficiency. For both assay approaches, the lowest detectable concentration of the synthetic target was 1 nmol/L (signal-to-noise ratio = 2), and the assays were linear up to 30 nmol/L.
The performance of the TruPoint-PCR and competitive TruPoint-PCR assays was compared by amplifying different amounts of DNA samples with wild-type, carrier, and mutant status for F508. Fig. 1
, C and D, shows representative results obtained with the end-point competitive TruPoint-PCR assay. After a 35-cycle PCR, the two assay approaches gave equivalent results and a detectable signal (signal-to-noise ratio = 2) with 1 ng of input DNA. Furthermore, DNA samples with wild-type, carrier, and mutant genotypes were correctly differentiated. The applicability of the TruPoint-PCR assay to real-time monitoring was also demonstrated. After 35 cycles, the real-time TruPoint-PCR assay gave detectable signal with 0.5–1 ng of input DNA (Fig. 1E
and Fig. 1 in the Data Supplement that accompanies the online version of this abstract at http://www.clinchem.org/content/vol50/issue10/).
In summary, the TruPoint-PCR and competitive TruPoint-PCR assays with dual-label TR-FQA allowed simultaneous detection of two alleles in one reaction, and the analysis of wild-type, carrier, and mutant DNA samples showed that the assays can specifically differentiate between these three genotypes. Although three to four probes are required for genotyping, the probes are relatively simple in design, including only one label moiety per probe. Furthermore, the lanthanide and quencher labels were directly coupled to the probes during solid-phase synthesis, allowing simple production of high-quality probes. Although direct comparison of TR-FQA PCR assays for different analytes is difficult, our results are comparable to those obtained by others. A TaqMan-like TR-FQA PCR assay has been used previously to detect input of 33 cDNA molecules (signal-to-noise ratio = 1.5) after 40 cycles of PCR (12). In addition, a competitive TR-FQA PCR assay has been used to genotype 50 ng of input DNA (signal-to-noise ratios ranging from 1.5 to 3.8) in a 40-cycle PCR protocol (13). We detected amplification product from at least 1 ng (equivalent to 330 target copies) of input DNA (signal-to-noise ratio = 2) when we used 35 cycles of PCR, which is practical for genotyping if the starting material is limited or if samples from various resources are used.
In conclusion, the TR-FQA technology allows enhanced homogeneous detection of amplification products. The assays developed are suitable for simplified genotyping, requiring only preparation of the amplification reactions, thermal cycling, and fluorescence measurement. The end-point measurement is the easiest way to record results, but the method also allows real-time monitoring. Furthermore, the closed-tube assay format eliminates contamination risk. The method is amenable to automation and suitable for the present instrumentation in many laboratories.
Acknowledgments
We thank Pirkko Grönroos, Satu Kling, Kirsi Nenonen, and Kristiina Raunio for excellent technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  P. von Lode, A. Syrjala, V. Hagren, H. Kojola, T. Soukka, T. Lovgren, and J. Nurmi Fully automated, homogeneous nucleic acid detection technology based on dry-reagent assay chemistry and time-resolved fluorometry. Clin. Chem., November 1, 2007; 53(11): 2014 - 2017. [Full Text] [PDF]
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