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Molecular mechanism controlling the incorporation of fluorescent nucleotides into DNA by PCR

✍ Scribed by Zhengrong Zhu; Alan S. Waggoner

Publisher
John Wiley and Sons
Year
1997
Tongue
English
Weight
140 KB
Volume
28
Category
Article
ISSN
0196-4763

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✦ Synopsis

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.

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