Example of Research

Basic outline of Ligation Independent Cloning.  Taken from Bitesize Bio article .

To study a specific gene in a host organism, it must first be inserted into a circular DNA molecule known as a plasmid vector which is compatible with the host’s molecular machinery.  This general process is known as cloning.  DNA cloning is traditionally accomplished through the use of DNA ligase, which is an enzyme that covalently combines the gene of interest with the plasmid molecule through the formation of phosphodiester bonds.  In principle, this biochemical technique is fairly straightforward and simple.  However, the combination of the gene with plasmid is typically a very inefficient procedure due to the in vitro (outside of the host cell) conditions under which the reaction occurs.  Cloning experiments conducted by students at Loras College have been particularly unsuccessful in the recent past.

There is an alternative method to traditional DNA cloning known as ligation-independent cloning (LIC).  This technique was first established in 1990 by Aslanidis and de Jong, and it utilizes multiple non-covalent interactions between complimentary strands of DNA (the gene and the plasmid vector) to circumvent the necessity of an in vitro ligation reaction.  This ligation step is unnecessary in ligation-independent cloning because the non-covalent interactions between the DNA strands are strong enough to allow the gene and plasmid to be successfully inserted into the host without dissociating from one another.  The host cell is then able to use its own DNA ligase to covalently combine the two pieces of DNA.  Once this is accomplished, the cell can begin to produce copies of the protein that gene codes for.

The original goal for my senior thesis project under the guidance of Dr. Kate Cooper was to create a ligation-independent cloning vector to be used for the study of the FCHO2 gene.  After more than a year of work, we were finally able to create this DNA molecule.  The next step of our project was to actually use the plasmid vector in ligation-independent cloning, but it proved difficult to find an established protocol.  The two protocols that I had access to gave contradictory advice, and neither of them gave positive results for the trials I conducted following their work.  Because of this, I was given the task of creating a protocol for the use of the LIC vector within Dr. Cooper’s research group.

I spent a large portion of my Intensive Research Experience J-term course trying to develop a reliable protocol for LIC.  This task was initially daunting because it was my first time performing research without any direct input from my research advisor.  I approached solving my problem by researching the ideal conditions to conduct the cloning reaction under.  However, this proved difficult because I was using an enzyme’s secondary function instead of the action it normally performs.  Therefore, I had to perform multiple trials trying to get my reactants in just the appropriate concentrations for LIC to be successful while trying to keep the protocol economical.  Also, alterations were made to make the protocol as time efficient as possible.  With a great deal of work and a little luck, I was able to produce an efficient and successful LIC protocol.

Since the creation of my LIC protocol, multiple other students, including myself, have used this procedure to successfully complete DNA cloning.  In fact, there has been more cloning accomplished at Loras College in the past month than in the previous five years combined.  It is rewarding to know that my work has directly assisted with the research of my peers and that it will continue to benefit students at Loras College for well into the future.

Reference:

         Aslanidis, C. and de Jong, P. J. (1990).  Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069 -6074.