can someone please explain DNA Sequencing?

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May 27, 2015
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I don't understand how just by the laser reading the fluorescent DDNTP terminator sequences, the DNA sequence is found? could someone explain please?

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Essentially, this method of DNA sequencing is made possible by the necessity of a free 3' -OH group for DNA polymerase to add nucleotides to a sequence.

In Sanger Sequencing (the name of this sequencing method), the same DNA sequence is used as a template in 4 separate PCR reactions. Each PCR reaction has a large amount of all four DNA nucleotides A, T, C, and G (which all contain a free 3' -OH group), which allows DNA replication to proceed normally. Each tube also contains a large amount of ONE dDNA (dideoxy-) nucleotide, which has no 3' -OH group. One tube contains dDNA T, one contains dDNA A, one contains dDNA C, and one contains dDNA G. DNA replication proceeds normally as is done in a PCR reaction, but some of the strands will stall out during each cycle due to the addition of a dDNA nucleotide, causing their length to be stunted. Thus, one reaction mixture with dDNA A nucleotides will contain DNA strands of many different lengths, each of which terminates with an A. The same is true for each different reaction mixture, each containing sequences that end with the designated dDNA nucleotide.

After the reaction is completed, each reaction mixture is individually loaded into the top of an electrophoretic gel. A current is applied to the gel so that DNA fragments migrate to the opposite end of the gel, with smaller fragments moving more quickly to the end of the gel. Because each reaction mixture is kept separate, bands in each lane indicate the last nucleotide in the DNA sequence of interest. For instance, if the band furthest to the bottom is found in the column that was loaded with the "T" reaction mixture, we know that the DNA elongation was halted due to the presence of a "T" dDNA nucleotide. T, then, is the first nucleotide of the sequence. If the next furthest band is in the "C" column, we know that the next nucleotide in the sequence is C, and so on.

There is a helpful video on YouTube called "DNA Sequencing: The Chain Termination Method (Sanger Sequencing)" that may be helpful as it provides a visual explanation. Feel free to reach out to me as well--it is hard to explain over text!

If I were you, though, I would focus more on test-taking strategy. As a general rule, the MCAT will provide super-detailed information like this in passages. If you know the basic science concepts and are familiar with the test and how to navigate it, you will be fine!

Good luck studying!