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372 Shen et al.
extended primers, which are the specific primers in the primer mix, would be allowed
during the termination/elongation because of the elevated temperature. If more template
DNA is available, fewer cycles may be used.
7. Depending on the length of labeled primers, the readable sequence will vary. For our
case of highly degenerate inosine-containing primers of p450 genes (see Subheading 1.
for a description of our p450 primers) a ladder from 25 bp downstream of the primer
was readable up to 300 bp.
8. A similar protocol of this method would be to omit one of the four dNTPs in the label step
and use at least one α- 35 S-labeled dNTP in the labeling mix. This will give an incomplete
elongation of the sequencing primer during the labeling step because the primer extension
will stop at the proper position when the omitted nucleotide is not present. The elongated
primers may be labeled if the labeled nucleotide is by chance present between the sequence
primer and the omitted nucleotide. This method is useful to sequence DNA when some
sequence information immediately downstream from the sequencing primer is available. In
such a case, one can decide which nucleotide to omit or to label in the label mix.
References
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2. Shen, Z., Wells, R. L., Liu, J., and Elkind, M. M. (1993) Identification of a cytochrome
p450 gene by reverse transcription-PCR using degenerate primes containing inosines. Proc.
Natl. Acad. Sci. USA 90, 11,483–11,487.
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and induction by aryl hydrocarbons of a murine cytochrome p450 gene, Cyplbl. DNA
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