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

368 Shen et al.
ladder. Also, direct sequencing of PCR products avoids the time-consuming cloning
of PCR products, and most of the available direct PCR sequencing protocols require
relatively small amounts of template.
Many protocols are available for the direct sequencing of PCR products. Most of
the protocols require specific sequencing primers. However, this means at least part of
the specific base sequence of the template is needed. This requirement may not be met
and, in many cases, information about the internal sequence of a gene may be lacking.
This shortcoming may apply to the PCR products of degenerate inosine-containing
primers of the cDNA of a new gene. Therefore, one may be forced to use the same
degenerate inosine-containing primers that were used in the PCR step for direct
sequencing. When primers have low degeneracy, they may be treated as sequencespecific
primers, and some of the direct-sequencing protocols, such as those described
in this volume, may be used with success. When only highly degenerate inosinecontaining
primers are available, these methods may not succeed.
To sequence a PCR product amplified via the use of a highly degenerate inosinecontaining
primers, several general factors must be kept in mind.
1. The sequencing primer(s) must anneal specifically to one site on the DNA fragment that is
to be sequenced, that is, a secondary annealing site must be avoided. Therefore, stringent
primer annealing temperatures are necessary.
2. A sufficient quantity of the specific primer should anneal to the correct site. Consequently,
the primer annealing temperature cannot be too high.
3. Reassociation of the double-stranded DNA template should be minimized. This requirement
generally can be met by using optimal PCR protocols for the sequencing reactions.
To perform the requirements above, a primer-labeling method, in which the primer
is labeled at the 5′ end, may be worth considering. Linear PCR is used to generate
the labeled dideoxynucleotide-terminated sequences (9,10). The use of this method
minimizes problems of template reassociation and/or mismatching of the primer
because the annealing time is relatively short. Also, the annealing temperature is higher
than it would be in most protocols that use DNA polymerases other than Taq, such as
T4 DNA polymerase, but the method requires a 5′ end-labeling step for which 35 S is
generally not suitable compared with 32 P because of its lower specific activity and the
lesser efficiency with which some enzymes label 5′ ends with α- 35 S ATP versus α- 32 P
ATP. Only 32 P can be used, even though its greater radiation hazard owing to its higher
β-particle emission and its shorter half-life make it less convenient. Furthermore,
when highly degenerate primers are used, higher primer concentrations in the reaction
mixture are needed to insure that sufficient specific priming will occur. The preceding
increases the hazard as well as the cost.
To assure that sequencing primer(s) anneal to a DNA template specifically, to
eliminate the need for 5′ end labeling, and to avoid reassociation of the double-stranded
DNA template, a two-step cycle-sequencing protocol is described to sequence products
amplified with degenerate inosine-containing primers. This method uses the same
degenerate primers that were used in PCR amplification. The method can be broken
down into two steps of linear PCR. The first step is for labeling the primers, and the
second is for the random dideoxy termination. As shown in Fig. 1, in the first step,
primers were extended and labeled with α- 35 S -dATP. The extension is limited and
performed under conditions of high stringency, low dNTP concentration, and a short