Design and optimization of molecular beacon real-time polymerase chain reaction assays.

During the last few years, several innovative technologies have become available for performing sensitive and accurate genetic analyses. These techniques use fluorescent detection strategies in combination with nucleic acid amplification protocols. Most commonly used is the real-time polymerase chain reaction (PCR). To achieve the maximum potential of a real-time PCR assay, several parameters must be evaluated and optimized independently. This chapter describes the different steps necessary for establishing a molecular beacon real-time PCR assay: (1) target design, (2) primer design, (3) optimization of the amplification reaction conditions using SYBR Green, (4) molecular beacon design, and (5) molecular beacon synthesis and characterization. The last section provides an example of a multiplex quantitative real-time PCR.

[1]  Sanjay Tyagi,et al.  Wavelength-shifting molecular beacons , 2000, Nature Biotechnology.

[2]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[3]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[4]  M. Wong,et al.  Rapid Identification of Candida dubliniensis Using a Species-Specific Molecular Beacon , 2000, Journal of Clinical Microbiology.

[5]  Fred Russell Kramer,et al.  Spectral Genotyping of Human Alleles , 1998, Science.

[6]  I G Wilson,et al.  Inhibition and facilitation of nucleic acid amplification , 1997, Applied and environmental microbiology.

[7]  T. B. Morrison,et al.  Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. , 1998, BioTechniques.

[8]  N. Thelwell,et al.  Duplex Scorpion primers in SNP analysis and FRET applications. , 2001, Nucleic acids research.

[9]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[10]  F. Kramer,et al.  Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis , 1999, Molecular microbiology.

[11]  C. Wittwer,et al.  Continuous fluorescence monitoring of rapid cycle DNA amplification. , 1997, BioTechniques.

[12]  Sanjay Tyagi,et al.  Multiplex detection of single-nucleotide variations using molecular beacons. , 1999, Genetic analysis : biomolecular engineering.

[13]  Sanjay Tyagi,et al.  Multiplex detection of four pathogenic retroviruses using molecular beacons. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. Syvänen,et al.  Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes. , 2000, BioTechniques.

[15]  Russell Higuchi,et al.  Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions , 1993, Bio/Technology.

[16]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[17]  Fred Russell Kramer,et al.  Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. , 2002, Nucleic acids research.

[18]  F. Kramer,et al.  Detection of Rifampin Resistance inMycobacterium tuberculosis in a Single Tube with Molecular Beacons , 2001, Journal of Clinical Microbiology.

[19]  F. Kramer,et al.  Thermodynamic basis of the enhanced specificity of structured DNA probes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Amalio Telenti,et al.  Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis , 1998, Nature Biotechnology.

[21]  H. Blom,et al.  Molecular beacons: a new approach for semiautomated mutation analysis. , 1998, Clinical chemistry.

[22]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[23]  H. Blom,et al.  Molecular beacons: colorful analysis of nucleic acids , 2002, Expert review of molecular diagnostics.

[24]  H Tanke,et al.  Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR. , 2001, Nucleic acids research.