Saturday, December 22, 2007

In the Lab 2: From Bird to DNA

To begin a phylogeography study, you need a large amount of genetic data. In my case, I am looking at differences between populations of Palm-Tanagers (Phaenicophilus) - see Part 1.

We get this data by collecting birds from a series of locales across the range of the two species. My study uses around five birds per locale, some studies use far more. Sites were varied, with some in lowlands, and some in the mountains. The intent was to get wide geographic spread and multiple types of topography represented in the samples, as well as to get samples across potential topographic barriers - the large mountain ranges on the island as well as the historic sea channels that once existed.

Bird DNA can be collected by two means. One method is lethal: the collection of a specimen, removal of blood and tissue for analysis, and deposition of a study skin in a museum. Many studies use this method. Although it may seem draconian to kill birds for study, museum specimens provide invaluable reference material for future research. The second method of collection is used in this project: the capture of a bird via mistnet and removal of a blood or feather sample. Avian red blood cells are enucleated, so a large amount of DNA can be recovered from a small blood sample or the tissue remaining in the base of a plucked feather. Museum skins as well can be a source of DNA, from small pieces of skin or toepad clippings. The nonlethal method is used in our work because some of our study birds are endangered, and the birds captured are also part of mark-recapture biodiversity studies.

Taking blood from a Chat-Tanager (Calyptophilus)
(Photo courtesy of Andrea Townsend)

Blood is collected by lightly piercing the vein along the wing with a small needle. A capillary tube is used to suck up the drop of blood created.The blood is transferred to a test tube and treated with blood lysis buffer. This buffer ruptures the blood cells, denatures proteins, and inactivates any enzymes that may denature and destroy the DNA. Thus, the DNA is safeguarded against decay and the blood may be stored in buffer at room temperature for long periods of time.

A blood sample

When all the blood samples required for a study are collected, the rest of the project is completed in the lab. For my study, all the field work was conducted before I joined the project. Thus, I was greeted by this wealth of data:

A whole lotta blood

The next step is to purify DNA from the blood sample. A small sample of the blood is treated with a variety of buffers and reagents, the end result being all of the non-DNA (proteins, membrane fragments, etc) are filtered out and purified DNA remains, as a clear viscous fluid.

Raw genetic material

DNA is purified from all of the blood samples and stored in a -20 degrees F freezer. This pure DNA is the raw material for study from which we can branch off in many directions of research.

On to Part 3: From DNA to Data

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