10) Amplified PCR fragments were purified using the Charge Switch PCR Clean Up Kit (Invitxoqen), quantified and then sequenced by the Sanger methodology using dideoxynucleotides
(ddNTPs) and the ABI PrismR BigDyeTM Terminator Kit and three pairs of primers that generated six sequences (three sense and three antisense) with approximately 500 base pairs overlapping.
Subsequent improvements in sequencing chemistry and methodology--such as incorporation of fluorophore-labeled dideoxynucleotides
for dye terminator sequencing (chain-termination method), cycled sequencing reactions catalyzed by thermostable DNA polymerases, automated capillary electrophoresis instruments, and laser detection methods--enhanced assay sensitivity.
Residual nucleotides were removed using ExoProStar 1-Step (GE Healthcare), and the PCR products were sequenced according to a standard protocol for fluorescently labeled dideoxynucleotides
(Applied Biosystems, Life Technologies) and separated on a capillary electrophoresis instrument (ABI 3500, Life Technologies).
SNaPshot is a commercially available kit for the multiplex detection of SNPs that relies on the extension of a primer annealed immediately adjacent to the SNP of interest, using fluorescently labeled dideoxynucleotides
This method is based on dye terminator chemistry, in which each of four dideoxynucleotides
is labeled with a different fluourochrome (Prober et al.
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.
Sequencing was performed by PCR techniques with 250 ng of plasmid DNA, following the protocols of Perkin-Elmer Applied Biosystems (Foster City, CA), using fluorescent-labeled dideoxynucleotides