Use of a new technique to map the porcine ? interferon gene to Chromosome 1 (original) (raw)

Mammalian Genome, 1994

Abstract

The polymerase chain reaction (PCR; Mullis et al. 1986) provides a means to amplify a single copy of DNA to literally hundreds of thousands of copies in a short time. It has been demonstrated that PCR can be performed on the surface of a slide containing cells (Yap and McGee 1991) and that the PCR process can incorporate biotin-11-dUTP into the amplification product (Weier et al. 1991). The objective of this investigation was to develop a method whereby the power of the PCR reaction could be coupled to physical gene mapping by amplifying single-copy genes in metaphase chromosome spreads, with non-isotopic detection methods to determine the site of amplification. The development of such a technique to place short, unique copy sequences that are PCR-based, such as expressed sequence tags (ESTs), microsatellites, and sequence-tagged sites (STSs), could considerably expedite the construction of gene maps of mammalian species. We describe here the application of this technique, which we have termed DISCPCR (Direct In-situ Single Copy) PCR to localize a short fragment of the porcine ~ interferon gene to a region of porcine Chromosome (Chr) 1 to which it has been previously placed by isotopic (Yerle et al. 1986) and nonisotopic (Sarmiento et al. 1993) in situ hybridization. Subsequent to the initiation of this investigation, a report appeared that further substantiates the feasibility of this approach; that report described a similar approach targeting repetitive human sequences (Gosden and Hanratty 1993). Metaphase chromosome spreads were prepared from pokeweed mitogen-stimulated lymphocytes of male pigs. The DNA sequence for the porcine o~ interferon gene (Lefevre and La Bonnardiere 1986) was used to design primers with the Primer 0.5 program (Whitehead Institute, Cambridge, Mass.). The forward primer sequence

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