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INTRODUCTION
This sequencing kit combines two revolutionary innovations for sequencing
DNA using radioactive labels. First, the label is incorporated into the DNA
sequencing reaction products by the use of four [α-
33
P]dideoxynucleotide
(ddNTP) terminators (G,A,T,C). The labeled ddNTPs are more efficient for
labeling sequencing experiments than other labeled nucleotides because they
specifically label only the properly terminated DNA chains. Also, since
prematurely terminated chains are not labeled, ‘stop’ artifacts and most
background bands are eliminated. As an additional benefit, the absence of
artifact bands allows the routine use of dITP, which can eliminate even very
strong compression artifacts.
The second innovation is the use of Thermo Sequenase DNA polymerase
‡
.
This enzyme has been engineered to efficiently incorporate dideoxynucleotides,
allowing the use of very low amounts of isotope ([α-
33
P]ddNTP) for the
termination reactions. Thermo Sequenase DNA polymerase is also
thermostable and performs very well in convenient and sensitive cycle or non-
cycle sequencing methods. This polymerase produces very uniform band
intensities (with dGTP), so mixed sequences (such as those of heterozygotes)
can be easily identified.
Thus, the kit offers:
• Clean, background-free sequences
• Complete elimination of compressions
• Efficient use of labeled nucleotides, less than 1µCi per sequence
• Convenient single-step protocol
• Uniform band intensities for identification of mixed sequences (
e.g.
heterozygotes)
• Sensitive cycle-sequencing protocols for sequencing 20fmol or less of
template
• Overnight exposures with ordinary autoradiography film—same day results
possible with fast films
• Exceptionally easy-to-read sequences
•
33
P for sharp autoradiogram resolution
• Sample storage for 1-2 days prior to running on gel