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Nucleic Acids Research, 1989, Vol. 17, No. 7 2517-2527
© 1989

MOLECULAR BIOLOGY

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA

Hartmut Voss, Christian Schwager, Ute Wirkner1, Brian Sproat, Jurgen Zimmermann, Andre Rosenthal2, Holger Erfie, Josef Stegemann and Wilhelm Ansorge*

European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg 1German Cancer Research Center, Experimental Pathology Im Neuenheimer Feld 280, 6900 Heidelberg, FRG 2Academy of Science of GDR, Institute of Molecular Biology GDR-1115 Berlin-Buch, GDR

*To whom correspondence should be addressed

Received February 15, 1989. Accepted March 1, 1989.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10–18 moles, corresponding to less than 106 molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5x104 copies of recombinant DNA.


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