Efficient PCR Amplification Research Articles

A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions.

For some time, gene disruptions in Candida albicans have been made with the hisG-URA3-hisG ('Ura-blaster') cassette, which can be re-used in successive transformations of a single strain after homologous excision of URA3. However, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product-directed gene disruptions. We describe here a gene disruption cassette, URA3-dpl200, with 200 bp flanking repeats that permit efficient PCR amplification. After transformation and integration to produce both arg5::URA3-dpl200 and rim101::URA3-dpl200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, respectively, can be obtained. We have used the cassette to create rim101::dpl200/rim101::URA3-dpl200 mutants exclusively through PCR product-directed disruption.

Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis

We have determined the nucleotide sequence of the rat hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8.) mRNA coding region and of adjacent, untranslated 5′ and 3′ mRNA, and we have designed an oligonucleotide primer pair for efficient PCR amplification of the rat hprt coding region. These sequence data and rat-specific primer pair will aid workers interested in coupling well-developed rat toxicologic and carcinogenicity bioassays with quantitative and molecular analyses of somatic mutation induction in rat cells in vivo and in vitro.