Identification and characterization of "zinc-finger" domains by the polymerase chain reaction.

AUTOR(ES)

Pellegrino, G R

RESUMO

We have developed a method for amplifying DNA fragments containing tandem arrays of "zinc-finger" sequences of the transcription factor IIIA (Cys2-His2) type by using the polymerase chain reaction. Because these sequences occur as tandem arrays, a ladder of bands is produced upon amplification using primers derived from the amino- and carboxyl-terminal sequences of a zinc-finger domain. The "rungs" of this ladder correspond to DNA fragments encoding one zinc-finger domain, two adjacent zinc-finger domains, and so on. This is demonstrated by isolating individual bands corresponding to n zinc-finger domains and reamplifying them with the same primers. This yields a band of the original size as well as bands corresponding to 1 through (n - 1) zinc-finger domains. Direct evidence that these bands encode zinc-finger domains was obtained by cloning and sequencing a collection of the amplification products. Due to the lack of redundancy in the sequences obtained, we conclude that each band corresponds to a large number of unique zinc-finger-encoding sequences. The results from amplification reaction mixtures using genomic DNA from a variety of sources as template provide further evidence that zinc-finger domains occur widely and frequently in eukaryotic genomes. We believe that this method is a powerful technique for the isolation and characterization of zinc-finger-encoding genes.

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