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Nucleic Acids Research, Vol 26, Issue 8 2031-2033, Copyright © 1998 by Oxford University Press

ARTICLES

Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms

A Fagotti, G Gabbiani, R Pascolini and P Neuville
Institute of Comparative Anatomy, University of Perugia, Perugia, Italy.

We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa.
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