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

Hartmut Voss | Zendy; Christian Schwager | Zendy; Ute Wirkner | Zendy; Brian S. Sproat | Zendy; Jürgen Zimmermann | Zendy; André Rosenthal | Zendy; Holger Erfie | Zendy; Josef Stegemann | Zendy; Wilhelm Ansorge | Zendy

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

Author(s) -

Hartmut Voss,

Christian Schwager,

Ute Wirkner,

Brian S. Sproat,

Jürgen Zimmermann,

André Rosenthal,

Holger Erfie,

Josef Stegemann,

Wilhelm Ansorge

Publication year - 1989

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/17.7.2517

Subject(s) - biology , primer (cosmetics) , microbiology and biotechnology , subcloning , recombinant dna , dna , oligonucleotide , genomic dna , multiple displacement amplification , dna nanoball sequencing , primer dimer , fluorescence , genomic library , dna sequencing , polymerase chain reaction , biochemistry , plasmid , gene , dna extraction , base sequence , chemistry , multiplex polymerase chain reaction , physics , organic chemistry , quantum mechanics

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.

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