Taq Polymerase 49of unknown sequences. This method is termed "anchor PCR" (75-77).
An alternative to eDNA synthesis is a combination of consecutive
RNA-DNA hybridization, S 1 nuclease digest, and PCR. This tech-
nique was applied successfully to trace CML-specific transcripts (78).
4.5. Sequencing of PCR Products
Sequencing PCR-amplified products is a time-saving procedure in
the analysis of genes. No time-consuming cloning of DNA is neces-
sary. On the contrary, errors made by Taq polymerase will lead to false
sequence information, if cloned PCR products are sequenced (3).
Sequencing revealed that, in 22 clones created from amplified DNA,
none had a faultless sequence (10). In a direct sequencing reaction of
amplified DNA, on the average, these shortcomings will not become
visible. Double-strand sequencing of a single copy gene was utilized
to typify mutations in the globin gene (79).
In order to increase the efficiency ot the sequencing reaction, diverse
strategies were developed aimed at yielding single-stranded DNA (80).
When biased primer concentrations are used, a phase of normal PCR will
precede a reaction producing dominantly single-stranded DNA (8,25). A
well-balanced ratio of the primer concentration is crucial. If the primer
concentration is too low, too little DNA will be available for single-strand
synthesis. On the other hand, the ratio of single-stranded to double-stranded
product will be inferior at primer concentrations that are too high. In
experiments working with asymmetric primer concentrations, the PCR
efficiency decreases to a factor of 1.7 as compared to amplifications with
stoichiometrlc concentrations.
Single-stranded DNA can be recovered by affinity chromatographic
purification after amplification with biotinylated primers (27). In an
elegant experiment (81), a phage promoter was attached 5' onto at least
one of the PCR primers. The amplified segments were transcribed into
RNA and sequenced with reverse transcriptase. Another way to gen-
erate single-stranded DNA involves a 5'-phosphorylated primer and
phage ~, exonuclease, which specifically attacks double-stranded DNA
only if there is a 5' terminal phosphate (82). With the dideoxy sequenc-
ing method, single copy genes can easily be sequenced with fluorescent
dye-labeled primers (29). As an alternative, Maxam-Gilbert sequenc-
ing of PCR products (83) or sequencing after incorporation of dNTPa~S
during the PCR is possible (18). When fluorescent dye-labeled prim-
ers are utilized, the cleavage products can be sequenced directly (28).