Functional cloning is a molecular cloning technique that relies on prior knowledge of the encoded protein’s sequence or function for gene identification. In this assay, a genomic or cDNA library is screened to identify the genetic sequence of a protein of interest. Expression cDNA libraries may be screened with antibodies specific for the protein of interest or may rely on selection via the protein function. Historically, the amino acid sequence of a protein was used to prepare degenerate oligonucleotides which were then probed against the library to identify the gene encoding the protein of interest. Once candidate clones carrying the gene of interest are identified, they are sequenced and their identity is confirmed. This method of cloning allows researchers to screen entire genomes without prior knowledge of the location of the gene or the genetic sequence. This technique can be used to identify genes that encode similar proteins from one organism to another. Similarly, this technique can be paired with metagenomic libraries to identify novel genes and proteins that perform similar functions, such as the identification of novel antibiotics by screening for beta-lactamase activity or selecting for growth in the presence of penicillin. Experimental workflow The workflow of a functional cloning experiment varies depending on the source of genetic material, the extent of prior knowledge of the protein or gene of interest and the ability to screen for the protein function. In general, a functional cloning experiment consists of four steps: 1) sample collection, 2) library preparation, 3) screening or selection and 4) sequencing. Sample collection Genetic material is collected from a particular cell type, organism or environmental sample relevant to the biological question. In functional cloning, mRNA is commonly isolated and cDNA is prepared from the isolated mRNA (RNA extraction). In certain circumstances genomic DNA may be isolated, particularly when environmental samples are used as the source of genetic material. Library preparation If the starting material is genomic DNA, the DNA is sheared to produce fragments of appropriate length for the vector of choice. The DNA fragments or cDNA are then treated with restriction endonucleases and ligated to a plasmid or chromosomal vectors. In the case of assays that screen for the protein or for its function, an expression vector is used to ensure that the gene product is expressed. The vector choice will depend on the origin of the DNA or cDNA to ensure proper expression and to