Open AccessDual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum
Author(s) -
Leyla Y. Bustamante,
Almudena Crooke,
Joaquín Martínez,
Amalia Díez,
José M. Bautista
Publication year - 2004
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/04363rn04
Subject(s) - plasmodium falciparum , biology , messenger rna , molecular beacon , genetics , stem cell , function (biology) , microbiology and biotechnology , computational biology , gene , immunology , malaria , oligonucleotide
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.