ensure that the encoded gene will fall within the limits of the vector’s insert size. The choice of host is important to ensure that the codon usage will be similar to the donor organism. The host will also need to guarantee that the proper post-translational modifications and protein folding will occur to enable proper functioning of the expressed proteins. Screening or selection The method of screening the prepared genomic or cDNA libraries for the gene of interest is highly variable depending on the experimental design and biological question. One method of screening is to probe colonies via Southern blotting with degenerate oligonucleotides prepared from the amino acid sequence of the query protein. In expression libraries, the protein of interest can be identified by screening with an antibody specific for the query protein via Western blotting to identify colonies carrying the gene of interest. In other circumstances, a specific assay can be used to screen or select for the protein’s activity. For example, genes conferring antibiotic resistance can be selected by growing the colonies of the library on media containing a specified antibiotic. Another example is screening for enzymatic activity by incubating with a substrate that is catalyzed to a colorimetric compound that can easily be visualized. Sequencing The final step of functional cloning is to sequence the DNA or cDNA from the clones that were successfully identified in the screen or selection step. The sequence can then be annotated and used for downstream applications, such as protein expression and purification for industrial applications. Advantages The advantages of functional cloning include the ability to screen for novel genes with desired applications in organisms that cannot be cultured, particularly from bacterial or viral specimens. Additionally, genes encoding proteins with related functions can be identified when there is low sequence similarity due to the ability to screen for the protein function alone. Functional cloning allows for gene identification without prior knowledge of the organism’s genome sequence or position of the gene within the genome. Limitations As with other cloning techniques, vector and host choice affect the success of gene identification via functional cloning due to cloning bias. The vector must have an insert size that will accommodate the entire DNA sequence of the expressed protein. Additionally, in expression vectors the promoters and terminators must function within the chosen host organism. The host choice may affect transcription and translation due to differing codon usage,