There are several
methods available to determine the actual sequence of nucleotides in a
segment of DNA. One procedure uses specially altered
nucleotides called dideoxynucloetides which have been made either
radioactive or fluorescent.
When enough of the targeted
DNA fragment (marker) has been amplified
mix is put through a series
sequencing reactions that are a variation of PCR. The amplified product from
the PCR is added to a reaction tube containing the same Taq DNA
polymerase used in the PCR, a primer that can hybridize at the desired
only one complementary strand of the DNA (as opposed to both
strands in PCR), and all four of the nucleotide bases (A, T, C, G.)
addition, small amounts of fluorescence labeled dideoxynucleotides (A,
are added to the mixture. Dideoxynucleotides are human-made
nucleotides whose sugar component is slightly different from that of the
nucleotides that make up DNA. (There is no OH on the
3' carbon.) Dideoxynucleotides can be picked up and added to a growing DNA chain.
However, as a result of this structural difference another nucleotide
cannot be added at its 3' end. Consequently, if one of the
dideoxynucleotides is added to a growing chain of nucleotides, the strand
will be terminated. Each dideoxynucleotide is labeled with a
different fluorescent compound so that it will give off an identifying
color in a laser beam.
20 - 30 cycles of the PCR heating and cooling, the resulting mixture
will contain a series of fragments of different lengths depending on how
many bases had been added to the chain before one of the dideoxynucleotides sneaked in and blocked further growth.
of billions of short fragments from the sequencing reactions is loaded
into glass capillary tubes that contain a gel solution that serves as a