Open AccessMolecular mechanism controlling the incorporation of fluorescent nucleotides into DNA by PCR
Author(s) -
Zhu Zhengrong,
Waggoner Alan S.
Publication year - 1997
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19970701)28:3<206::aid-cyto4>3.0.co;2-b
Subject(s) - primer (cosmetics) , primer dimer , microbiology and biotechnology , fluorescence , dna , dna polymerase , cyanine , nucleotide , polymerase , primer extension , polymerase chain reaction , biology , template , chemistry , biochemistry , multiplex polymerase chain reaction , base sequence , nanotechnology , gene , materials science , physics , organic chemistry , quantum mechanics
The efficiency and yield of incorporation of fluorescent nucleotides into DNA by polymerase chain reaction (PCR) have been investigated with linear amplification (PCR with single‐stranded template and single primer). In the present study, we prepared single‐stranded templates with defined sequences and used dUTP attached to the fluorescent label with linkers of different lengths. Incorporation and yield of the modified dUTP were reduced when the sequence demanded that multiple dyes be inserted at adjacent sites. The interactions between the polymerase and cyanine‐labeled sites on the extending strand probably terminated the chain extension. Thus, because labeling density was increased, the yield of PCR was reduced. We also found that the interactions between the primer and dye‐labeled sites on template disturb primer annealing and lead to a decrease in PCR yield. Cytometry 28:206–211, 1997. © 1997 Wiley‐Liss, Inc.