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DNA Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #37)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00


INTRODUCTION
DNA measurements are important for many aspects of molecular biology. Of thespectrophotometric methods available, UV absorbance measurement at 260 nm isthe simplest and has recently gained widespread popularity in the microplateformat.1 However, A260 measurements do not distinguish between dsDNA,ssDNA and RNA. Also, they are relatively insensitive (limit of quantitationapproximately 250 ng/mL in traditional spectrophotometers2,3 and approximately 100 ng/well in a 96-well microplate1). Below these levels, analternative method must be used such as the PicoGreen reagent from Molecular Probes, Inc.4 The latter reagent binds to dsDNA with high affinity and becomesfluorescent. The limit of detection in a microplate well is approximately 50 pgdsDNA per well.4 Recently, a luminometric method intended for use in standardtube luminometers was introduced by Promega Corporation.5 The DNAQuant is specific for linear dsDNA (including PCR fragments) and is not affected bycontaminants (e.g. ssDNA , RNA, protein) which interfere with other methods.This method has been adapted to Molecular Devices Lmax microplateluminometer with a resultant lower limit of approximately 5 pg DNA per well.The method and results are described below.


PRINCIPLE OF
ASSAY
The DNAQuant uses a coupled enzyme system to produce ATP in an amount proportional to the amount of dsDNA present, followed by addition of luciferin/luciferase reagent to generate light in proportion to the amount of ATP (Figure 1).The coupled set of reactions consists of a pyrophosphorylation andtransphosphorylation (pyro/transphosphorylation). The pyrophosphorylationreaction uses T4 DNA polymerase in the reverse
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