A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides
PCR reactions, in the presence of the different dideoxynucleotides
, are performed as described above.
(ddUTP, ddGTP, ddCTP, and ddATP) were added to terminate elongation (Sambrook & Russell 2001).
Sanger Sequencing, also called Sanger Dideoxy Sequencing, is the controlled interruption of enzymatic DNA replication through the use of dideoxynucleotides
in primer-directed DNA extension to produce discrete DNA fragments (11,41) (Fig.
Solid phase capturable dideoxynucleotides
for multiplex genotyping using mass spectrometry.
Nucleotide sequences were determined by oligonucleotide-directed dideoxynucleotide
chain-termination sequencing reactions utilizing 300 ng of template DNA, forward (M13F) and reverse (M-13R) primers, fluorescently-labeled dideoxynucleotides
and AmpliTaq FS DNA polymerase in a cycling sequencing method.
Then a mixture of all four dideoxynucleotides
, each coupled to a different fluorescent dye, are added and primer extension performed.
15 U of thermosequenase, and a mixture of dideoxynucleotides
(ddATP, ddCTP, and ddTTP) and the deoxynucleotide dGTP (64 [micro]mol/L each).
The cost of manufacturing primers with 3' modified locked nucleic acids is high, however, and primer with added 3' dideoxynucleotides
is not widely available commercially, so this expertise-requiring modification must be performed in the laboratory.
Primer-extension techniques use labeled or unlabeled dideoxynucleotides
to terminate the amplification in a sequence-dependent manner (10).
In our case, we have demonstrated that the C3 linker is indeed an excellent choice for such a blocking agent, but alternatives may include other carbon spacers and 3'terminal dideoxynucleotides
, although the latter would add substantially to synthesis costs.
Our limited data (not shown) indicated that the extension products were retained in the order of C < G < T < A, which represent the 3' dideoxynucleotides
of the products.