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Nucleic Acids Research, 2003, Vol. 31, No. 10 2636-2646
© 2003 Oxford University Press

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA

Taurai Tasara, Bernhard Angerer, Martine Damond, Holger Winter, Sabine Dörhöfer, Ulrich Hübscher1 and Mario Amacker

Gnothis SA, PSE-B, EPFL, CH-1015 Lausanne, Switzerland and 1 Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (VentR exo–) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, VentR exo–, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.


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