Homologous and heterologous FISH painting with PARM-PCR chromosome-specific probes in mammals

Abstract

Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30 degrees C during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.