使用PCR进行荧光DNA标记

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使用PCR进行荧光DNA标记

Fluorescent DNA Labeling by PCR
Introduction
Amplification of DNA using the polymerase chain reaction
(PCR)* provides the opportunity to label the resulting product
with either modified nucleotides or oligonucleotide primers.
Incorporation of fluorescent labels offers the advantages of direct
detection, sensitivity, and multicolor capability. Both fluorescently
labeled deoxynucleotide triphosphates (dNTPs) and fluorescently
end-labeled oligonucleotide primers are available for use in PCR
product labeling.
Fluorescent dNTP Incorporation
Cy3-dCTP and Cy5-dCTP
As a model PCR system, a 268-bp fragment of the human b-globin
gene was amplified. Each dNTP was used at a final concentration
of 50 mM in these reactions with labeled dCTP mixed in different
proportions with unlabeled dCTP. The labeled nucleotide was
used at concentrations of 2.5 mM, 5 mM, 10 mM, 16.5 mM, 25 mM,
33.5 mM, and 50 mM. Control reactions were also carried out with
no added label. PCR products were resolved in 10% polyacrylamide
gels. Cy?3 labels were detected with the FluorImager? 595 and
Cy5 labels were imaged using the Storm? 860 (Figure 1). Two
parameters were determined from these gels. First, the relative
amount of fluorescent label incorporated in each amplified
product was quantified. Second, overall PCR yield was estimated
by staining gels with either SYTO? 59 from Molecular Probes or
?Vistra Green from Amersham PhARMacia Biotech UK Limited.
See Table 1.

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