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 NEW!

Hammerhead High School Research Site

 
Adapted from Compagno’s 1984 FAO guide to sharks of the world, this map shows the expected distribution of scalloped hammerhead sharks, Sphyrna lewini.

We are attempting to collect samples from throughout the Pacific Ocean, and now have samples from Panama, Taiwan, and Australia. We would very much like to get samples from Pacific Islands.

Only small pieces of shark tissue are needed for genetic analysis. Here are two pieces of shark fin, which were clipped from the tip of the pectoral fin with a hole punch.

After collection, tissues are placed in 95% ethanol for preservation. Here a fin clip has been removed from the preservative, subsampled, and ‘minced’ to prepare for the extraction of DNA. The tissue samples are finely chopped in order to expose more surface area and to speed digestion. Here are three vials of tissue that have been ‘minced’ and then lysed with proteinase k, a special digestive enzyme. After digestion, the tissues are liquid and can be pipetted away from the hard, calcareous dermal denticles that also make up the shark skin. The digested tissue is then filtered through a special silica membrane, which binds the DNA and isolates it from the other tissue components.

After   DNA is extracted, its concentration is measured using a spectrophotometer  and   the DNA is visualized on an agarose gel. The gel is put in an electrophoresis chamber where electrical currents separate particles by size (smaller ones move faster). In this picture, individual DNA samples running horizontally. Those with lots of large, high quality of DNA have bright bands on the left hand side of the photo.

Using a process called polymerase chain reaction (PCR), primers (special pieces of DNA) bind to and cut the sample DNA at specific places. An extension enzyme, polymerase, then aids in replication of the target fragment. The end result of the PCR process is  many copies of the target fragment, in our case the d-loop portion of the mtDNA. In order to determine if the PCR process has been successful, the target fragments are visualized on an agarose gel. In the photo you can see the band of various samples, all consisting fragments a little larger than 1000 bases (as compared with the 100 pair ladder scale at the top of the photo).

The last step in the process is to clean and then sequence the base pairs of the target fragment. We are using automated sequencing, which produces a printout of the base pairs making up the target fragment like that shown to the left.

 

 

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This site was created and is maintained by Timothy Fitzgerald
Last updated September 17, 2004 10:23 PM HST