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DNA sequencing reactions all use a primer
to initiate DNA synthesis. This primer will determine the starting point
of the sequence being read, and the direction of the sequencing reaction.
Most DNA sequencing reactions use dideoxy nucleotides (ddNTP) to stop DNA synthesis at specific nucleotides. For example, if the ddCTP to the right is incorportated into a growing strand of DNA, the lack of a free 3´ OH group would prevent the next nucleotide from being added, and the chain would terminate.
In automated sequencing we use a different fluorescent label attached to each of the four dideoxy nucleotides (ddA, ddC, ddG and ddT). Thus we can determine the terminal base in each fragment of DNA.
The two animations below illustrate how DNA sythesis and dideoxy termination are used to sequence DNA. |
