Limits...
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.

Show MeSH
Detection of SNP variations using tetranucleotide-long Angler probes. Shown are the scatter plots for three SNPs found in human genomic DNA. The polymorphism numbers as well as the abbreviated probe structures are listed on the top of each diagram. The SNP-discriminating nucleotides are underlined. FAM, HEX and YY are abbreviations of the fluorescent dyes used in the study. Q is a quenching dye, whereas R is a 3′-anchoring sequence. Full sequences of the probes and the primers used in each SNP case are listed in Table 2 and also illustrated in Supplementary Figure S12. No target controls (NTC), the homozygous (homo) and heterozygous (hetero) signal groups are marked by a specific color. All experimental details can be found in ‘Materials and Methods’ section and Table 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814350&req=5

gkt782-F5: Detection of SNP variations using tetranucleotide-long Angler probes. Shown are the scatter plots for three SNPs found in human genomic DNA. The polymorphism numbers as well as the abbreviated probe structures are listed on the top of each diagram. The SNP-discriminating nucleotides are underlined. FAM, HEX and YY are abbreviations of the fluorescent dyes used in the study. Q is a quenching dye, whereas R is a 3′-anchoring sequence. Full sequences of the probes and the primers used in each SNP case are listed in Table 2 and also illustrated in Supplementary Figure S12. No target controls (NTC), the homozygous (homo) and heterozygous (hetero) signal groups are marked by a specific color. All experimental details can be found in ‘Materials and Methods’ section and Table 1.

Mentions: A SmartCycler® instrument (Cepheid, Sunnyvale, CA, USA) was used in the proof-of-principle studies and qPCR experiments (Figures 3 and 4). Single nucleotide polymorphism (SNP) genotyping (Figure 5 and Supplementary Figures S9–S11) was conducted on a plate-based Mx3000P QPCR System of Agilent Technologies (Santa Rosa, CA, USA). To ensure reproducibility of the data, the experiments were repeated at least twice. The real-time curves shown in Figures 3 and 4A represent an average of 4–5 identical reactions performed simultaneously. In all studies, background fluorescence was subtracted using instrumental software, and the data, i.e. fluorescence versus PCR cycle, were transferred to an Excel file (Microsoft Office) for further processing and threshold calculations.


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

Kutyavin IV - Nucleic Acids Res. (2013)

Detection of SNP variations using tetranucleotide-long Angler probes. Shown are the scatter plots for three SNPs found in human genomic DNA. The polymorphism numbers as well as the abbreviated probe structures are listed on the top of each diagram. The SNP-discriminating nucleotides are underlined. FAM, HEX and YY are abbreviations of the fluorescent dyes used in the study. Q is a quenching dye, whereas R is a 3′-anchoring sequence. Full sequences of the probes and the primers used in each SNP case are listed in Table 2 and also illustrated in Supplementary Figure S12. No target controls (NTC), the homozygous (homo) and heterozygous (hetero) signal groups are marked by a specific color. All experimental details can be found in ‘Materials and Methods’ section and Table 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt782-F5: Detection of SNP variations using tetranucleotide-long Angler probes. Shown are the scatter plots for three SNPs found in human genomic DNA. The polymorphism numbers as well as the abbreviated probe structures are listed on the top of each diagram. The SNP-discriminating nucleotides are underlined. FAM, HEX and YY are abbreviations of the fluorescent dyes used in the study. Q is a quenching dye, whereas R is a 3′-anchoring sequence. Full sequences of the probes and the primers used in each SNP case are listed in Table 2 and also illustrated in Supplementary Figure S12. No target controls (NTC), the homozygous (homo) and heterozygous (hetero) signal groups are marked by a specific color. All experimental details can be found in ‘Materials and Methods’ section and Table 1.
Mentions: A SmartCycler® instrument (Cepheid, Sunnyvale, CA, USA) was used in the proof-of-principle studies and qPCR experiments (Figures 3 and 4). Single nucleotide polymorphism (SNP) genotyping (Figure 5 and Supplementary Figures S9–S11) was conducted on a plate-based Mx3000P QPCR System of Agilent Technologies (Santa Rosa, CA, USA). To ensure reproducibility of the data, the experiments were repeated at least twice. The real-time curves shown in Figures 3 and 4A represent an average of 4–5 identical reactions performed simultaneously. In all studies, background fluorescence was subtracted using instrumental software, and the data, i.e. fluorescence versus PCR cycle, were transferred to an Excel file (Microsoft Office) for further processing and threshold calculations.

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