Dideoxy sequencing method

The basic idea is to be able to stop synthesis of a complementary DNA strand at various points and look at the terminator nucleotide of complementary strands of different lengths. This is done by culturing the DNA strand of interest with two types of nucleotides, normal DNA nucleotides which use deoxyribose and special DNA nucleotides which use dideoxyribose instead of deoxyribose as the five carbon sugar. When DNA is synthesized, synthesis proceeds starting at the 3' end of the original strand. New nucleotides are added by attaching the deoxyribose by the hydroxyl group at the 3' position of the deoxyribose molecule. Dideoxyribose has a hydrogen in place of the hydroxyl group so synthesis stops when a dideoxyribose containing nucleotide ends up opposite a complementary base in the original DNA strand.

To sequence DNA using this technique scientists culture copies of a DNA strand to be sequence with all four normal nucleotides, DNA polymerase and one of the four possible dideoxyribose nucleotides. Synthesis proceeds until a complementary nucleotide with dideoxyribose added to the growing complementary strand, at which point synthesis stops. The result is a series of complementary DNA strand of different lengths depending on when in the synthesis process the dideoxyribose nucleotide comes in by chance. To actually sequence the DNA all the scientist has to do in theory is to culture the original DNA strand in for separate cultures using the four normal nucleotide bases but with only one of the four dideoxyribose nucleotides. Then subject the sets of fragments to electrophoresis. This yields bands that travel different distances from the starting point and the sequence of bands corresponding to each terminal dideoxyribose nucleotide are read off as shown in the figure.

This Figure shows partial results for a hypothetical sequence of nucleotide bases serving as a template. Note the primer is a short sequence of nucleotides called an oligonucleotide which is complementary to a region of the template just in front of the part of the DNA to be sequenced. The primer is needed because the DNA polymerase needs to recognize the 3' hydroxyl group of an already attached oligonucleotide in order for synthesis of the complementary strand to proceed.

The resulting bands allow the scientist to read off the sequence of terminal dideoxyribose containing nucleotides as shown. Notice that the short fragments travel furthest in the electrophoresis set up and the fragment closest to the bottom then which has the dideoxyribose nucleotide containing the base cytosine is just that fragment in our example. Hence C is the first base in the complementary strand, A is the next base and so forth. Thus the entire sequence of nucleotides in the complementary strand can be direct read off the electrophoresis and the original strand is obviously the complement.

For the following results of electrophoresis, what is the sequence of nucleotide bases in the original DNA used as the template?

The whole process can be automated, the bandings being detected by spectrophometry and the results analyzed by computer software. When sequencing the DNA of a whole chromosome, the DNA is first cultured with restriction enzymes to produce various sized fragments, each fragment is sequenced and then computer searches are conducted to piece the sequences into the proper order by looking for overlaps.