iPBS: a universal method for DNA fingerprinting and retrotransposon isolation.
Molecular markers are essential in plant and animal breeding and biodiversity applications, in human forensics, and for map-based cloning of genes. The long terminal repeat (LTR) retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their "copy and paste" life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Nevertheless, up to a year has been required to develop a retrotransposon marker system in a new species, involving cloning and sequencing steps as well as the development of custom primers. Here, we describe a novel PCR-based method useful both as a marker system in its own right and for the rapid isolation of retrotransposon termini and full-length elements, making it ideal for "orphan crops" and other species with underdeveloped marker systems. The method, iPBS amplification, is based on the virtually universal presence of a tRNA complement as a reverse transcriptase primer binding site (PBS) in LTR retrotransposons. The method differs from earlier retrotransposon isolation methods because it is applicable not only to endogenous retroviruses and retroviruses, but also to both Gypsy and Copia LTR retrotransposons, as well as to non-autonomous LARD and TRIM elements, throughout the plant kingdom and to animals. Furthermore, the inter-PBS amplification technique as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence knowledge.
Kalendar
Ruslan
R
MTT/BI Plant Genomics Laboratory, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, P.O. Box 65, Helsinki, Finland.
Antonius
Kristiina
K
Smýkal
Petr
P
Schulman
Alan H
AH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D044822:Biodiversity; D019295:Computational Biology; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism; D016678:Genome / Q000235:genetics; D027845:Malus / Q000235:genetics; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D018626:Retroelements / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics; D003313:Zea mays / Q000235:genetics
D017931:DNA Primers; D018626:Retroelements