Dynamic Analysis of Viral Populations by Direct DNA Sequencing and Solid-Phase Technology

The improvements in DNA-sequencing technology in recent years has opened new possibilities, from large scale genome projects to routine diagnostic applications. Among the most important developments towards automated systems has been the introduction of the polymerase chain reaction (PCR) and the replacement of isotopic labels by fluorescent dyes and on-line monitoring of the DNA sequence. The use of PCR for amplification of genetic material allows for direct DNA sequencing, which avoids both time-consuming cloning steps as well as control sequencing necessary to exclude “mutations” introduced by the Taq DNA polymerase (1 ,2 ). Thus the DNA sequence of a sample can be determined rapidly and will represent the sequence of the sample prior to amplification, as the Taq polymerase errors will not significantly contribute to the resulting signal. Different methods can be employed to obtain labeled dideoxy DNA fragments according to the Sanger methodology (3 ), such as, using fluorescent-labeled primers, dideoxy chain terminators, or fluorescentlabeled dATPs. These possibilities enables many different flexible non-radioactive sequencing alternatives. In principle all commercial instruments are based on the excitation of sequencing bands by a laser beam which then can be detected in the gel during electrophoresis. An important ability with the commercially available instruments is that these enable reliable quantitation of polymorphic and heterozygous positions using different software tools (4 ,5 ).