Functional cloning
[1][2][3] In this assay, a genomic or cDNA library is screened to identify the genetic sequence of a protein of interest.
This method of cloning allows researchers to screen entire genomes without prior knowledge of the location of the gene or the genetic sequence.
In general, a functional cloning experiment consists of four steps: 1) sample collection, 2) library preparation, 3) screening or selection and 4) sequencing.
Genetic material is collected from a particular cell type, organism or environmental sample relevant to the biological question.
[7] 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.
[5] Another example is screening for enzymatic activity by incubating with a substrate that is catalyzed to a colorimetric compound that can easily be visualized.
[10] 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.
[11] In model organisms such as mice or yeast, this method is used more frequently as the information about the position of a gene of interest can be obtained from the sequenced genome.
Gibson assembly is a quick cloning method that uses three primary enzymes; 5' exonuclease, polymerase and ligase.
[17] While this method isn't strictly an alternative, it does allow the movement of DNA fragments from one plasmid to another quicker than creating a whole new genomic library.
[17] This can come in handy if an individual wants to try functional cloning in a wide range of bacteria to try to combat the issue with codon bias.
Metagenomics studies all the genetic material from a specific environmental sample, such as the gut microbiome or lake water.
Some novel proteins that have been discovered from marine environments include enzymes such as proteases, amylases, lipases, chitinases, deoxyribonucleases and phosphatases.
[20] One could set up a functional cloning screen to find homologous polymerases that have the added advantage of being thermostable at high temperatures.
[21] The enzyme was discovered using functional cloning from a viral host originally found in Octopus hot springs (93 °C) in Yellowstone National Park.
[22] Using a functional cloning-based technique, DNA isolated from human microflora were cloned into expression vectors in Escherichia coli.
[22] If a plasmid contained a gene insert that provided antibiotic resistance the cell survived and was selected on the plate.
[22] Based on selection of cell colonies that survived, a better picture of genetic factors contributing to antibiotic resistance were pieced together.
[22] By using a functional cloning-based technique one is able to elucidate genes giving rise to antibiotic resistance to better understand treatment for bacterial infections.
