Question

Illustrate steps to show the construction of a recombinant DNA molecule.

Hint:

Remember the key steps involved in making a recombinant DNA molecule. Understand the role of restriction enzymes, DNA ligase and host organisms in this.

Answer 1

The construction of a recombinant DNA molecule generally involves the following steps: Isolation of DNA: The first step is to isolate the desired DNA fragment from a source (such as genomic DNA) and also the vector DNA (usually a plasmid) that will carry the desired DNA sequence into a host organism. Cutting the DNA: Both the target DNA fragment and the vector DNA are then cut using the same restriction enzyme. This enzyme will create “sticky ends" or specific sequence on the DNA that can be used to join the target and the vector DNA. Ligation: Next, the target and vector DNAs are joined together using a DNA ligase. This enzyme facilitates the formation of phosphodiester bonds, which joins the DNA sequences together, resulting in a recombinant DNA molecule, also known as a ‘chimeric’ DNA. Transformation: This new molecule (vector plus insert) is then introduced into a host organism like E.coli} through the process called transformation. Here the bacteria can grow, and it will replicate the vector containing the insert DNA. Selection and Screening: The transformed cells are then screened to verify whether the DNA fragment has been successfully inserted. This usually involves antibiotic resistance and other selection techniques.