Research Trends in Biochemical Analysis

Lai, Edward P. C.

doi:10.4172/2161-1009.1000e144

Cited by 1 publication

(1 citation statement)

References 22 publications

(22 reference statements)

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“…The decrease of the amplitude of the FCS curve during DNA cleavage by restriction of enzyme Hind III agrees well with the dsDNA double labeling efficiency [5]. Compared with the previous method developed in our earlier study, this method shows big improvements in the calculation of the double labeling efficiency [5,17]. The previous method is not flexible and requires exactly twice as high concentration of the dsDNA in the reference sample.…”

Section: Resultssupporting

confidence: 77%

A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

et al. 2014

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We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value.

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Section: Resultssupporting

confidence: 77%