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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. |
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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. |
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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). |
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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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population genetics main page
This site was
created and is maintained by Timothy Fitzgerald
Last updated September 17, 2004 10:23 PM HST
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