Direct genomic fluorescent on-line sequencing and analysis usingin vitroamplification of DNA

Abstract

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 5×10⁴ copies of recombinant DNA.