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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

Authors :
Almudena Crooke
Amalia Diez
Leyla Y. Bustamante
José M. Bautista
Joaquín Martínez
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.

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