CGDP - DNA Extraction

Extraction Amplification Sequencing

 

 
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FLMNH 2005


 
DNA extraction involves breaking down cellular membranes, deactivating enzymes that chew up DNA, and stabilizing the DNA in a hospitable buffer. The DNA is separated from the other tissue components into a usuable form for further amplification. We usually extract DNA from foot muscle tissue, but have had success extracting DNA from the heart, gonads, eggs, radular ribbon, and mantle. Only a small amount of tissue is required, usually about the size of one pinhead.

Once the DNA is extracted, now referred to as Genomic DNA, we check the quantity and quality of the extraction by visualizing a standard amount on an agarose gel. DNA is negatively charged, and when it encounters and electric current, it runs toward the positive side. Smaller pieces of DNA migrate faster through the gel than larger pieces. This way we can separate the DNA from lower molecular weight to higher molecular weight. Good extractions ("genomic DNA") is almost entirely high molecular weight. This results from quick, fresh preservation and extraction. DNA can be sheared, or "chewed up" into smaller pieces, without proper preservation. This happen quickly after the organism dies. Formalin preserved material is extremely sheared, with no high molecular weight material remaining. Alcohol preserved material tends to also be sheared.

High molecular weight material will almost always yield successful amplifications. In fact, amplification was successful for the all extractions visualized in this gel except #701 which was from a dried preserved animal of C. irrorata. Amplification even worked for #723 although from the gel, the outlook didn't look promising. Technically, it takes only one copy for for the amplification to work because of its exponential nature.

Extractions can be performed both in the field and the laboratory. Once the DNA is extracted, we usually store it frozen until it is used for amplification via PCR.