Dual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum.
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.
Bustamante
Leyla Y
LY
Universidad Complutense de Madrid, Madrid, Spain.
Crooke
Almudena
A
Martínez
Joaquín
J
Díez
Amalia
A
Bautista
José M
JM
D020856:AT Rich Sequence / Q000235:genetics; D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D009862:Online Systems; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism*; D015347:RNA Probes / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D015347:RNA Probes; D012333:RNA, Messenger; D014157:Transcription Factors