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使用PCR进行荧光DNA标记

2009-1-13 18:34:13 3237

0 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. f0WZXsfV.pdf (165.48 KB, 下载次数: 18 ) 0
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