John M. S. Bartlett.pdf - Bio-Nica.info

Inosine-Containing Primers 369
Fig. 1. Procedure of cycle sequencing with degenerate inosine-containing primers. Two
linear PCR steps are involved. 1. Label PCR uses low dNTP concentrations, a low temperature,
and short times for primer annealing/elongation to produce incomplete extension of specific
primers. As a result, specific primers are labeled and extended. The extended and labeled
primers have a higher melting temperature than the native printers. 2. Termination PCR using
a higher annealing/elongation temperature and is performed with higher dNTP concentrations
and in the presence of ddNTPs. Only the extended and labeled primers are involved in the
termination reaction.
interval so that the specific primer in the mixture is favored and a limited length of
primer extension is achieved. In the second step, dideoxynucleotide terminations are
effected at a more stringent elevated annealing/elongation temperature. The result is
that only the extended and labeled primers enter into the termination reactions.
We have used this method to sequence amplified cytochrome p450 cDNA fragments
with a highly degenerate inosine-containing primer (1,2). In our case, a set of degenerate
primers was used to amplify a presumably novel cytochrome p450 gene(s). The upstream
sense primer was a mix of 192, 20mer, containing three inosines that theoretically could
anneal to 12,288 different sequences. The downstream antisense primer was a mix of
144, 23 mer, containing five inosines or 147,456 different possible sequences.
In what follows, we will only describe the sequencing reactions. Procedures for
sequencing gel electrophoresis can be found in Chapter 51.
2. Materials
1. A thermal cycler: Cetus Perkin–Elmer Model 480 (see Note 1).
2. PCR tubes (0.5 mL).