LA-PCR-based quick method for the identification of genes responsible for the complementation of Saccharomyces cerevisiae mutations.

Cloning of Saccharomyces cerevisiae genomic DNAs that complement yeast mutations includes (i) screening of genomic DNA library clones by complementation and (ii) identification of the DNA regions essential for the complementation ability. Since commonly used yeast genomic DNA libraries made on YEp-type (2) or YCptype (6) plasmids harbor about 10 kb of yeast chromosomal DNA, the inserted yeast DNA usually contains several genes. Frequently, to identify the candidate gene on a plasmid essential for the complementation, subcloning analysis is carried out. However, this step is laborious, dependent on the physical map, and sometimes time consuming. Transposon mutagenesis also is used, but with either method, one has to know the physical map of the inserted DNA in advance. The current protocol was developed in order to replace the subcloning step during cloning of yeast genes. It consists of screening transposon-mutagenized plasmids that do not complement the mutation and determination of the position of transposon insertions with the LA (long and accurate) polymerase chain reaction (PCR) method (1,3). LA-PCR is applicable here because even the entire 10-kb DNA fragment can be amplified with this technique. Direct sequencing of the LA-PCR products makes it possible to determine the DNA sequence of the cloned genes. Bacterial transposition, combined with the LA-PCR method, allows easy identification of complementing yeast genes without any restriction enzyme digestion. To show the utility of this protocol, I demonstrate here mapping of the yeast calmodulin gene, CMD1, on a pST26 plasmid (Figure 1), which was isolated from a YEp13-based genomic library (10).