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Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction.

Kutyavin IV - Nucleic Acids Res. (2013)

Bottom Line: The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends.The unparalleled cost-effectiveness of the inventory approach is discussed.Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

View Article: PubMed Central - PubMed

Affiliation: Perpetual Genomics, Woodinville, WA 98077, USA.

ABSTRACT
Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends. Hybridization of this sequence to a complementary 'anchoring' tail introduced at the 3' end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a 'universal' probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

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Shown are PCR primers, FRET probes and the detected β2-microglobulin gene sequence used in the experiments of Figures 3 and 4. Wherever it is possible, the oligonucleotides are aligned with the target sequence in 5′→3′ orientation as indicated. The primers’ flap sequences participating in the amplicon folding and their binding sites in the detected DNA are underlined. All nucleosides in the probe-anchoring sequence R were modified with 2-amino deoxyadenosine, 5-propynyl deoxyuridine and 5-propynyl deoxycytidine. FAM is 6-fluorescein and BHQ1 is a Black Hole Quencher 1.
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gkt782-F1: Shown are PCR primers, FRET probes and the detected β2-microglobulin gene sequence used in the experiments of Figures 3 and 4. Wherever it is possible, the oligonucleotides are aligned with the target sequence in 5′→3′ orientation as indicated. The primers’ flap sequences participating in the amplicon folding and their binding sites in the detected DNA are underlined. All nucleosides in the probe-anchoring sequence R were modified with 2-amino deoxyadenosine, 5-propynyl deoxyuridine and 5-propynyl deoxycytidine. FAM is 6-fluorescein and BHQ1 is a Black Hole Quencher 1.

Mentions: The PCR reaction mixtures were prepared by mixing concentrated stock solutions of the reagents at room temperature. All assays were conducted in an aqueous buffer of 20 mM Tris–HCl (pH 8.0) containing 50 mM KCl and a mixture of all four dNTPs (200 µM each). The FRET probes (Taqman and Angler) were always applied at 200 nM concentration. The concentrations of other key reaction components such as forward and reverse primers, magnesium chloride, JumpStart™ Taq DNA polymerase varied depending on the study and method used. These variations including the PCR cycle profiles are listed in Table 1. Human genomic DNA (GenScript, Piscataway, NJ, USA) was used as a target for detection of the β2-microglobulin gene (GenBank accession #NM004048) in the amounts as indicated in each particular experiment (Figures 3 and 4). Taqman PCR was conducted using the reaction composition and cycle profile previously optimized for this assay (18). Sequences of the primers and probes used in the proof-of-principle (Figure 3) and qPCR experiments (Figure 4) are shown in Figure 1. The final reaction volume in these experiments (SmartCycler) was 25 µl.Figure 1.


Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction.

Kutyavin IV - Nucleic Acids Res. (2013)

Shown are PCR primers, FRET probes and the detected β2-microglobulin gene sequence used in the experiments of Figures 3 and 4. Wherever it is possible, the oligonucleotides are aligned with the target sequence in 5′→3′ orientation as indicated. The primers’ flap sequences participating in the amplicon folding and their binding sites in the detected DNA are underlined. All nucleosides in the probe-anchoring sequence R were modified with 2-amino deoxyadenosine, 5-propynyl deoxyuridine and 5-propynyl deoxycytidine. FAM is 6-fluorescein and BHQ1 is a Black Hole Quencher 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814350&req=5

gkt782-F1: Shown are PCR primers, FRET probes and the detected β2-microglobulin gene sequence used in the experiments of Figures 3 and 4. Wherever it is possible, the oligonucleotides are aligned with the target sequence in 5′→3′ orientation as indicated. The primers’ flap sequences participating in the amplicon folding and their binding sites in the detected DNA are underlined. All nucleosides in the probe-anchoring sequence R were modified with 2-amino deoxyadenosine, 5-propynyl deoxyuridine and 5-propynyl deoxycytidine. FAM is 6-fluorescein and BHQ1 is a Black Hole Quencher 1.
Mentions: The PCR reaction mixtures were prepared by mixing concentrated stock solutions of the reagents at room temperature. All assays were conducted in an aqueous buffer of 20 mM Tris–HCl (pH 8.0) containing 50 mM KCl and a mixture of all four dNTPs (200 µM each). The FRET probes (Taqman and Angler) were always applied at 200 nM concentration. The concentrations of other key reaction components such as forward and reverse primers, magnesium chloride, JumpStart™ Taq DNA polymerase varied depending on the study and method used. These variations including the PCR cycle profiles are listed in Table 1. Human genomic DNA (GenScript, Piscataway, NJ, USA) was used as a target for detection of the β2-microglobulin gene (GenBank accession #NM004048) in the amounts as indicated in each particular experiment (Figures 3 and 4). Taqman PCR was conducted using the reaction composition and cycle profile previously optimized for this assay (18). Sequences of the primers and probes used in the proof-of-principle (Figure 3) and qPCR experiments (Figure 4) are shown in Figure 1. The final reaction volume in these experiments (SmartCycler) was 25 µl.Figure 1.

Bottom Line: The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends.The unparalleled cost-effectiveness of the inventory approach is discussed.Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

View Article: PubMed Central - PubMed

Affiliation: Perpetual Genomics, Woodinville, WA 98077, USA.

ABSTRACT
Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends. Hybridization of this sequence to a complementary 'anchoring' tail introduced at the 3' end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a 'universal' probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

Show MeSH