Multiplex–microsphere–quantitative polymerase chain reaction: Nucleic acid amplification and detection on microspheres

We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a “universal” oligonucleotide “tagged” polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the...

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Published inAnalytical biochemistry Vol. 432; no. 1; pp. 23 - 30
Main Authors Liang, Fang, Lai, Richard, Arora, Neetika, Zhang, Kang Liang, Yeh, Che-Cheng, Barnett, Graeme R., Voigt, Paul, Corrie, Simon R., Barnard, Ross T.
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LanguageEnglish
Published United States Elsevier Inc 01.01.2013
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Abstract We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a “universal” oligonucleotide “tagged” polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex–microsphere–quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.
AbstractList We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.
Author Barnard, Ross T.
Arora, Neetika
Barnett, Graeme R.
Corrie, Simon R.
Lai, Richard
Yeh, Che-Cheng
Liang, Fang
Zhang, Kang Liang
Voigt, Paul
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Issue 1
Keywords Polymerase chain reaction
Microsphere
Quantitative
Real time
Luminex
Multiplex
Language English
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Snippet We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a “universal”...
We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal"...
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SubjectTerms detection limit
fluorescent dyes
Fluorescent Dyes - chemistry
gene amplification
genes
influenza
Limit of Detection
Luminex
Microsphere
Microspheres
Multiplex
Multiplex Polymerase Chain Reaction
Neisseria
Nucleic Acid Amplification Techniques
nucleic acids
oligonucleotides
Polymerase chain reaction
Quantitative
quantitative polymerase chain reaction
Real time
Title Multiplex–microsphere–quantitative polymerase chain reaction: Nucleic acid amplification and detection on microspheres
URI https://dx.doi.org/10.1016/j.ab.2012.09.017
https://www.ncbi.nlm.nih.gov/pubmed/23000310
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