CGDP - DNA Sequencing
Part 1

Extraction Amplification Sequencing

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FLMNH 2005


 
   The first step after PCR is to clean up the newly created products. The extra primers, unincorporated G's, A's, T's and C's, salts and buffers need to be removed so that only the target pieces of DNA remain. For this research we use a resin that binds the DNA pieces in combination with a filter. First, the unwanted ingredients are flushed through the filter, but the DNA remains behind because it is attached to the resin which cannot pass through. Then the DNA is freed from the resin by adding water. Because the DNA is no longer bound to the resin, it passes through the filter and is collected for sequencing. Again, the DNA is run out on an agarose gel to make sure it is clean and to estimate its concentration for sequencing. This gel shows cleaned PCR products ready for sequencing from two different genes, COI and 16S. The COI gene is slightly longer and runs a little slower than the 16S pieces.
   The image to the right is a cartoon of how sequencing works. Similar to the intial PCR, the target pieces are cycled through the denaturation, annealing and extension phases. However, this time while most pieces get copied over and over again like in regular PCR, some strands get stopped as they extend from the primers. A special terminating G, A, T, or C is incorporated that does not permit further extension. Done over 25 cycles, eventually pieces of DNA are made that have been stopped at every single nucleotide site. This is called cycle sequencing. After 25 cycles the tubes contain millions of variable length DNA strands - covering all the possible lengths of the target gene. The same process is done in another tube from the other primer side, so that two complementary DNA sequences can be generated and compared for accuracy. In the past the DNA strands incorporated radioactivity so they would expose X-ray film. Now special fluorescent dyes are used like in the image to the right, a different color for each of the four different terminating nucleotides.
   Now that the cycle sequencing is complete, how do we go from the liquid in the little tubes and see what the sequence looks like? Well, again we run out the reactions on gels, but instead of agarose gels, this time we use more sensitive sequencing gels.