The chapter describes a detection method that is based on the sequence-dependent hybridization of... more The chapter describes a detection method that is based on the sequence-dependent hybridization of fluorogenic reporter molecules called “molecular beacons’’ and its applications. Molecular beacon probes represent a new class of oligonucleotides that can hybridize and report the presence of specific nucleic acids in homogeneous solutions. In addition, improvisations on the basic theme of molecular beacons have also appeared in the literature. Molecular beacons are primarily employed as highly specific amplicon detection probes in homogeneous, real-time, multiplex gene amplification assays. On the other hand, the generation of all five fluorescent colors during PCR amplification indicates that the mycobacteria in the sample are rifampin susceptible. Infection by certain types of human papillomavirus (HPV) can lead to cervical cancer; therefore, accurate identification of these oncogenic HPV genotypes is critical. One assay detects HPV-DNA by SYBR Green® and then distinguishes the seven most prevalent high-risk HPV genotypes by using real-time molecular beacon PCR. They can be used as hybridization detection probes, not only for real-time monitoring of DNA amplification in vitro but also for real-time monitoring of the distribution and transport of mRNAs in living cells. The current available applications of molecular beacons for detection of pathogenic microorganisms are listed in the chapter. Efforts are being made to explore their applications in many areas including genotyping of infectious agents and mutation analysis for the identification of drug-resistant pathogens.
The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in... more The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.
The chapter describes a detection method that is based on the sequence-dependent hybridization of... more The chapter describes a detection method that is based on the sequence-dependent hybridization of fluorogenic reporter molecules called “molecular beacons’’ and its applications. Molecular beacon probes represent a new class of oligonucleotides that can hybridize and report the presence of specific nucleic acids in homogeneous solutions. In addition, improvisations on the basic theme of molecular beacons have also appeared in the literature. Molecular beacons are primarily employed as highly specific amplicon detection probes in homogeneous, real-time, multiplex gene amplification assays. On the other hand, the generation of all five fluorescent colors during PCR amplification indicates that the mycobacteria in the sample are rifampin susceptible. Infection by certain types of human papillomavirus (HPV) can lead to cervical cancer; therefore, accurate identification of these oncogenic HPV genotypes is critical. One assay detects HPV-DNA by SYBR Green® and then distinguishes the seven most prevalent high-risk HPV genotypes by using real-time molecular beacon PCR. They can be used as hybridization detection probes, not only for real-time monitoring of DNA amplification in vitro but also for real-time monitoring of the distribution and transport of mRNAs in living cells. The current available applications of molecular beacons for detection of pathogenic microorganisms are listed in the chapter. Efforts are being made to explore their applications in many areas including genotyping of infectious agents and mutation analysis for the identification of drug-resistant pathogens.
The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in... more The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.
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