IRAP and REMAP for retrotransposon-based genotyping and fingerprinting.
Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.
Kalendar
Ruslan
R
MTT/BI Plant Genomics Laboratory, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, P.O. Box 56, Viikinkaari 4, FIN-00014 Helsinki, Finland.
Schulman
Alan H
AH
D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel / Q000379:methods; D018745:Genome, Plant; D005838:Genotype; D018895:Microsatellite Repeats; D015336:Molecular Probe Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D018626:Retroelements
D017931:DNA Primers; D018626:Retroelements