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Synthesis of Highly Modified DNA by a Combination of PCR with Alkyne-Bearing Triphosphates and Click Chemistry

Authors

  • Johannes Gierlich,

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
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  • Katrin Gutsmiedl,

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
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  • Philipp M. E. Gramlich,

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
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  • Alexandra Schmidt,

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
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  • Glenn A. Burley Dr.,

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
    2. Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK, Fax: (+44) 116-252-3789
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  • Thomas Carell Prof.

    1. Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstrasse 5–13, Haus F, 81377 Munich, Germany, Fax: (+49) 89-2180-77756
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Abstract

We report the combination of “click chemistry” with PCR by using alkyne-modified triphosphates for efficient and homogeneous labeling of DNA. A series of modified PCR products of different lengths (300, 900, and 2000 base pairs) were prepared by using a variety of alkyne- and azide-containing triphosphates and different polymerases. After intensive screening of real-time PCR methods, protocols were developed that allow the amplification of genes by using these modified triphosphates with similar efficiency to that of standard PCR. The click reaction on the highly modified PCR fragments provided conversion rates above 90 % and resulted in the functionalization of hundreds of alkynes on large DNA fragments with superb selectivity and efficiency.

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