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-33P]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-33P]ddNTP) for the termination reactions. Thermo Sequenase DNA polymerase is also thermostable and performs very well in convenient and sensitive cycle or noncycle 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 1∝Ci 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
•33P for sharp autoradiogram resolution
•Sample storage for 1-2 days prior to running on gel