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 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.