Manipulating the genome
Molecular cloning produces identical, recombinant DNA molecules for downstream applications, such as gene expression and mutational analysis.
The process begins with nucleic acid isolation, which can include plasmid DNA, genomic DNA, or total RNA. Alternatively, readymade clones for your gene of interest, containing the cDNA or ORF, are available to skip this step. If the initial DNA quantity is low, PCR amplification of template material may be required.
DNA samples may undergo restriction enzyme digestion and gel analysis to detect loci of interest. The DNA is extracted from the gel, purified, and modified so that its free ends do not anneal with each other, but remain accessible to the plasmid during ligation. The plasmid behaves as the vector to get the DNA into a bacterial cell.
E. coli or yeast species are typically used for transformation, the process of picking up extracellular DNA, usually a plasmid with the gene insert.
Host machinery can replicate and express the desired gene product, while the cell multiplies and creates exponential copies of the recombinant DNA contained within the bacterial plasmid. Screen and selection assays identify which bacterial colonies contain the gene of interest. These clones can be further manipulated for gene expression assays, protein purification.
The Fundamental Cloning Workflow
- Preparation of starting material
- DNA fragment generation
- DNA fragment purification
- Modification of DNA ends
- Analysis of recombinant clones