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Synthesis of Highly Modified DNA by a Combination of PCR with Alkyne‐Bearing Triphosphates and Click Chemistry
Author(s) -
Gierlich Johannes,
Gutsmiedl Katrin,
Gramlich Philipp M. E.,
Schmidt Alexandra,
Burley Glenn A.,
Carell Thomas
Publication year - 2007
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200700502
Subject(s) - click chemistry , alkyne , combinatorial chemistry , dna , chemistry , azide , polymerase chain reaction , surface modification , nucleotide , selectivity , organic chemistry , biochemistry , gene , catalysis
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.