Designing a Cloning Scheme

Overview

Cloning a gene is similar to finding a needle in a hay stack. A gene, even in the simplest of organisms, represents only a tiny fraction of the DNA in a genome (all the genetic information in the cell). A typical scheme for cloning involves removing the genomic DNA from a donor, breaking the DNA into many thousands of small pieces, and then searching those pieces for the desired gene. The search is accomplished by using molecular probes that adhere specifically to the targeted gene.

Strategies for cloning DNA differ markedly from those used for isolating and purifying proteins. Cloning relies on using nucleic acid probes to search for specific DNA or on introducing genome fragments into new hosts in which the targeted gene is expressed. Probes are used to locate DNA while expressed genes are usually identified by phenotypic changes in the host. Cloning does not always start with DNA. Messenger RNA (mRNA) can be isolated and converted into complementary DNA (cDNA). As we will see, cloning from either DNA or RNA each has merit depending on the circumstances.

The first half of this manual focused on the production, purification, and analysis of α-galactosidase. The final product of the purification, which was isolated by native PAGE, would have been subsequently sequenced. The resulting amino acid sequence is the data necessary to link the protein to the gene that codes for α-galactosidase.

This chapter will provide an overview to the strategies and approaches used in cloning. A major strategy for cloning applies protein sequence data, and this is the strategy demonstrated in this manual. The laboratory exercise will emphasize the most important aspect of experimentation, namely planning and strategy.