Taq Polymerase% faultless Sequences
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[log] AmplificationFig. 7. Dependence of error rate and fragment length.In a technique referred to as differential PCR, alterations of genes
can be monitored when two genes are coamplified in one reaction
vessel. In this method, the primers are competing for the limiting
enzyme; therefore, gene copy numbers can be estimated semiquanti-
tatively even in the plateau phase of the PCR (89).
Quantitation of mRNA transcription in different cell types was
successfully performed using Southern hybridization to an internal
standard and to the amplified product (90). Monitoring antiviral therapy
benefits from the exceptional sensitivity of the PCR as well (91).
A test system where standards identical to the target sequence, except
for one restriction site, are coamplified provides an elegant way for the
absolute determination of mRNA amounts. Consequently, both sig-
nals are enhanced to the same extent; variations in the reaction effi-
ciency will not influence the result. After completion of the PCR, the
standard is cleaved and compared with the target sequence (43). An
alternative to blotting techniques is the utilization ofbiotinylated prim-
ers; the PCR products are immobilized on avidin agarose and hybrid-
ized to specific probes (14).
- Sequencing with Taq Polymerase
Although Taq polymerase is considered often to be only useful for
the PCR, last but not least, it should be pointed out that the enzyme is
ideal for dideoxy sequencing, the reasons being: