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Dual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum
Dual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum
- Source :
- BioTechniques. 36:488-494
- Publication Year :
- 2004
- Publisher :
- Future Science Ltd, 2004.
-
Abstract
- The genome of the human malaria parasite Plasmodium falciparum is extremely AT-rich such that it is particularly difficult to design standard probes to identify and quantify specific transcripts. Biased AT genome contents (70%–80%) lead to a high proportion of short repetitions and a low free energy of binding between target sequences and their specific probes during hybridization. This causes nonspecific annealing and high background noise. We constructed molecular beacon probes with dual-function stems to avoid nonspecific detection and establish identical melting patterns for use with several fluorescent probes for the analysis of mRNA expression in P. falciparum in real-time reverse transcription PCR (RT-PCR) assays. The method proved highly efficient at detecting low transcript levels in P. falciparum microcultures. Conditions were established for two types of real-time instruments, demonstrating that molecular beacons with dual-function stems are a useful tool for the functional analysis of high AT genomes. The procedure could be adapted to high-throughput gene expression protocols for the biomolecular screening of the P. falciparum and other AT-rich genomes.
- Subjects :
- Genetics
biology
Reverse Transcriptase Polymerase Chain Reaction
Gene Expression Profiling
Plasmodium falciparum
RNA Probes
biology.organism_classification
AT Rich Sequence
Online Systems
Genome
General Biochemistry, Genetics and Molecular Biology
Reverse transcription polymerase chain reaction
Apicomplexa
Molecular beacon
parasitic diseases
Gene expression
Animals
Protozoa
Parasite hosting
RNA, Messenger
Transcription Factors
Biotechnology
Subjects
Details
- ISSN :
- 19409818 and 07366205
- Volume :
- 36
- Database :
- OpenAIRE
- Journal :
- BioTechniques
- Accession number :
- edsair.doi.dedup.....fbba0e1f04b5b74a8b36d4dc872d8206
- Full Text :
- https://doi.org/10.2144/04363rn04