Authors
Heather Etchevers Ph.D.
Summary
The dideoxynucleotide or dye termination method for sequencing uses synthetic nucleotides that lack the -OH at the 3′ carbon atom. This sort of dideoxynucleotide can be added to the growing DNA strand; when it is, chain elongation stops because there is no 3′ -OH to which to attach the next nucleotide. If the ratio of normal nucleotide to the dideoxy versions is high, some DNA strands will succeed in adding hundreds of nucleotides before insertion of the dideoxy version halts the process. After DNA polymerization, the fragments are separated by length on a capillary sequencer. The resolution is sufficient for a difference of one nucleotide to separate that strand from the next shorter or next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser; a scanner and software order and interpret the sequence for further analysis.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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