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使用PCR进行荧光DNA标记

2009-1-13 18:34

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