Cloning our way to a perfect sequence

Kelsey Coates, Duquesne University

The Approach: In my first blog post, “FROM FEMALE TO MALE – MUD SNAILS TELL ALL!,” I described the goal of my research, to sequence isoforms of a hormone receptor called the Retinoid X Receptor (RXR) in the eastern mud snail.  

Mud snails all over a beach at Fort Johnson, SC.

These isoforms have yet to be sequenced in the mud snail! But what exactly is a DNA sequence? DNA is made of building blocks called nucleotides. A DNA sequence is the order of the nucleotides. A sequence like ACG could tell the organisms’ body to do one thing while a sequence like AGC could tell the organisms’ body to do another. A bit of the sequence has already been identified, but there is a gap in the sequence we are still trying to figure out.  

Theoretically, different chemicals or different concentrations of the same chemical can change the relative levels of the RXR isoforms. If this hypothesis is confirmed, mud snails can be used in the future to detect contaminants that affect marine organisms in the Charleston Harbor. Their patterns of isoform expression might suggest which seasonal contaminants are present in the environment where they live. For example, chemical one may trigger isoform A which has sequence ACG while chemical two may trigger isoform B which has sequence AGC.            

So how will we get these sequences? It starts with amplifying the known sequence of the mud snail that surrounds the isoform, including the mysterious gap. Amplification will be done by polymerase chain reaction (PCR) to ensure there are thousands of copies of the DNA to work with. After purification, the sequence is ready to be incorporated into a plasmid along with an antibiotic resistance component. Bacteria, like E. Coli, store their DNA in plasmid form compared to the double-helix form of humans.

Plates of E.Coli in the presence of Ampicillin set in the incubator.

Luckily for us, plasmids are easily manipulated and are reproduced rapidly in bacteria. E. Coli will be grown in the presence of the antibiotic ampicillin with the sequence we cloned into its DNA.  If the sequence is incorporated into the plasmid, the bacteria will have anti-biotic resistance and be able to grow on the ampicillin plates. The bacterial colonies with our plasmid will be PCR amplified. Then, after a final plasmid preparation, the samples from E. Coli can be sent to a lab that specializes in sequencing. Hopefully the lab will identify the gap and we will achieve our goal!

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Demetri Spyropoulos, Edwina Mathis, Dr. Bob Podolsky, The Fort Johnson REU Program, The Hollings Marine Lab, NOAA, and The Grice Marine Lab. This research was supported by the Fort Johnson REU Program, NSF DBI-1757899.

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