MOLECULAR MECHANISM CONTROLLING THE INCORPORATION OF FLUORESCENT NUCLEOTIDES INTO DNA BY PCR

The efficiency and yield of incorporation of fluorescent nucleotides i nto DNA by polymerase chain reaction (PCR) have been investigated with linear amplification (PCR with single-stranded template and single pr imer), In the present study, we prepared single-stranded templates wit h defined sequences and used dUTP attached to the fluorescent label wi th linkers of different lengths. Incorporation and yield of the modifi ed dUTP were reduced when the sequence demanded that multiple dyes be inserted at adjacent sites. The interactions between the polymerase an d cyanine-labeled sites on the extending strand probably terminated th e chain extension. Thus, because labeling density was increased, the y ield of PCR was reduced. We also found that the interactions between t he primer and dye-labeled sites on template disturb primer annealing a nd lead to a decrease in PCR yield. (C) 1997 Wiley-Liss, Inc.