Synthesis of highly modified DNA by a combination of PCR with alkyne-bearing triphosphates and click chemistry.
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.
Gierlich
Johannes
J
Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Germany.
Gutsmiedl
Katrin
K
Gramlich
Philipp M E
PM
Schmidt
Alexandra
A
Burley
Glenn A
GA
Carell
Thomas
T
D000480:Alkynes / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics*; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D013053:Spectrophotometry; D044366:Transition Temperature
D000480:Alkynes; D010710:Phosphates; D011743:Pyrimidines; D012333:RNA, Messenger; D004247:DNA; C030986:pyrimidine