【摘要】 chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. this is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. probes developed in this manner can be used to investigate chromosome homologies in closely related species. here we describe a protocol recently used in the gymnotiform species group eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.
【关键词】 fluorescence in situ hybridization chromosome painting sex chromosomes crossfish
chromosome-specific sequences are highly desired in studies focusing on comparative genomics and genomic organization. the two most straightforward paths for obtaining chromosome-specific makers are flow-sorting, commonly referred to as facs ( fluorescence activated cell sorting ), and chromosome microdissection. in flow-sorting, chromosomes are sorted using a laser system that distinguishes chromosomes depending on their size and fluorochrome affinity (at, gc base content) while in chromosome microdissection chromosomes or chromosomal segments are literally scraped and collected.
it is generally assumed that flow-sorting generates paints of greater complexity and coverage because this method allows the collection of massive amounts of chromosomes (around 300-500) in a highly automated procedure (ferguson-smith et al., 1998). however, the costs of equipment and need of high quality cell cultures make this technique not suitable for most fish cytogenetics laboratories. specifically concerning genetic studies in fish, a new difficulty arises, since most fish species present a highly uniform karyotype regarding chromosomal size and base content.