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

AUTOR(ES)

Voss, H

RESUMO

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 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.

Documentos Relacionados

• Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.
• On-line integration of PCR and cycle sequencing in capillaries: from human genomic DNA directly to called bases
• High speed on-line DNA sequencing on ultrathin slab gels.
• Coupled amplification and sequencing of genomic DNA.
• Direct sequencing of enzymatically amplified human genomic DNA.