Detection of a single-locus gene on channel catfish chromosomes by In- Situ polymerase chain reaction

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An in-situ polymerase chain reaction (ISPCR) procedure was applied to chromosomal localization of the gene, Ig H, encoding the immunoglobulin heavy chain of channel catfish (Ictalurus punctatus). Metaphase chromosomes were prepared by a replication banding procedure and subjected to ISPCR using biotin-labeled primers. The hybridization signals were detected with an avidin-fluorescein isothiocyanate (FITC)-based method, and chromosome bands revealed by simultaneous or sequential treatment methods. Standard fluorescent in-situ hybridization (FISH) was performed on chromosome preparations to compare with the ISPCR procedure. The Ig H gene was detected at the telomeric position of a chromosome with a relative length of 3.2 ± 0.2%. The Ig H-bearing chromosome detected by the FISH method was identical to that found by ISPCR procedure. Visibility of chromosome bands was reduced by heat and salt treatments and could not be analyzed after thermocycling. Therefore, specific identity of the chromosome bearing the Ig H gene remains unknown. Banding of fish chromosomes is difficult and poses a barrier for applying current molecular techniques to physical mapping of teleost genomes. Application of the ISPCR to chromosomal mapping is new for fish species and is only in initial stages of development for higher vertebrates.

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Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology

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