Gap Repair

This is a method frequently used to recover a specific sequence from the chromosome onto an episomal plasmid. Gap repair utilizes the host cell's recombination/ repair and DNA replication machinery to fill an artificially created deletion in a

URA3

Figure 1.5 Gap repair homologous sequence carried on the plasmid. Its most common use is for cloning different alleles of a cloned gene.

One starts with the DNA fragment of interest cloned into a plasmid vector that is maintained as an extrachromosomal element, such as YRp or YEp. Using restriction endonucleases that cut sites in the insert fragment but not in vector sequences, one creates a deletion internal to the yeast DNA fragment. It is essential to leave at least 50 bp of insert fragment at either end to provide homology to the chromosomal site as a substrate for recombination. This linearized and gapped fragment is then transformed into the host cell and transformants are selected using the marker gene carried by the plasmid vector. For the example shown in Figure 1.5, the host strain is ura3 and repair of the gap is essential if the cell is to maintain the plasmid and to be able to grow on a selection medium lacking uracil. The gapped region is filled by a gene-conversion-like event between the gapped plasmid and the homologous chromosomal site. The arrows in Figure 1.5 indicate the endpoints of the gap and the positions where the exchange events will initiate. The free ends of the gapped fragment invade the homologous chromosomal sequence, DNA replication of the gapped region occurs from these ends using the chromosomal sequence as template, and the gap is filled.

Gap repair is used to recover different alleles of the cloned sequence from the chromosome. For example, one has cloned the wild-type allele of a gene and wants to clone the available mutant alleles. Another use of gap repair is in fine structure mapping of recessive mutant alleles. If a mutation maps outside the gapped region, then filling in the gapped region of the wild-type allele carried on the plasmid with the chromosomal sequence will result in the restoration of the wild-type allele on the plasmid copy of the gene and stable transformants with the wild-type phenotype of the gene of interest will result. If the mutation maps within the gap, then only stable transformants with the mutant phenotype will be obtained.

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