A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Abstract

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10 7 initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

“…In this work we used a different quantitative real-time PCR assay based on the use of environmentally sensitive lanthanide chelates and the 5Ј-3Ј exonucleolytic activity of the DNA polymerase (25). In addition, the quantitative real-time PCR was conducted with a quencher oligonucleotide and a dual-temperature assay (26).…”

High-Throughput Genetic Analysis Using Time-Resolved Fluorometry and Closed-Tube Detection

“…Because terbium signals can be measured by timeresolved fluorometry, we can routinely use approximately 10 -100 times less probe in our assays, which further diminishes assay costs. Finally, these labels can be easily combined with conventional fluorescent markers with a short decay time without the need to rely on any spectral resolution software, since the label signals are temporally separated (10). This enables for example the use of intercalating dyes to control the amplification efficiency obviating the need to use consensus probes to eliminate false negative results caused by unsuccessful amplification.…”

“…The probes were labeled with an isothiocyanate-modified, fluorescent, environment-sensitive terbium chelate (2,2Ј,2Љ,2ٞ-{{6,6Ј-{4Љ-[2-(4-isothiocyanatophenyl)ethyl]1H-pyrazol-1,3-diyl}bis(pyridine)-2,2Ј-diyl}bis(methylenenitrilo)}tetrakis(acetato) terbium-(III); Perkin-Elmer Life Sciences Wallac, Finland) as described previously (10). Briefly, 5Ј-amino-modified oligonucleotide probes were incubated with a 100-fold molar excess of the chelate in 50 mM sodium carbonate buffer (pH 9.8) at 37°C overnight.…”

Detection of Fungicide Resistance

“…PCR based detection of fungicide resistance depends upon the ability of the reaction to selectively amplify specific regions of DNA, and usually require several post-PCR steps, including agarose gel electrophoresis for either confirmation of amplicon presence or size, or restriction enzyme analysis (Lesniak et al 2011;Fontaine et al 2009;Quello et al 2009; and reviewed by Ma and Michailides 2005). The development of new fluorescent techniques (LAMP, etc) has led to novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acid sequences (Nurmi et al 2000) and allow for the detection of a specific PCR product in a homogeneous solution without the need to open the amplification tubes after PCR or gel electrophoresis (Tomlinson et al 2012). In these techniques and their variations, PCR products are monitored as they are generated during the course of the reaction via one of two ways: By fluorescent or chemiluminescent dyes that bind to double-stranded DNA in a nonspecific fashion, or by fluorescence-labeled probes that bind to specific sequences.…”

“…Furthermore, the results can be read in real time as the PCR product accumulates or at the end of the thermal cycling protocol directly from the amplification wells. Although many scientists believe that the choice between real time or "standard" PCR (and gel electrophoresis) depends on whether a quantitative or qualitative assay is desired (Nurmi et al 2000), the reality is that equipment expense and laboratory expertise limits most "applied" labs, resulting in a preponderance of scientifically dazzling techniques that may be used in a human clinical setting, or a plant pathology laboratory focused on basic science, but are rarely, if ever, subsequently tested using isolates from a field failure.…”