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Evaluation of FlashPlate in a Helicase Assay

Abstract

This study demonstrated the efficacy of the FlashPlate as a high throughput screening (HTS) platform for a helicase functional assay. Helicase activity was measured on a streptavidin coated FlashPlate (SMP103), using two DNA oligos. One of the oligos was biotinylated; the other was 5' end-labeled with [33P]-γ-ATP. The oligos were preannealed to each other, to form a [33P]-biotinylated DNA duplex substrate, then immobilized on the SMP103 FlashPlate. The FlashPlate platform provided an effective and economical basis for the evaluation of DNA helicase enzyme activities.

Introduction

Helicases are enzymes that catalyze the unwinding of DNA strands, promoting replication and playing an important role in DNA repair. Conventional helicase functional assays require that unwound DNA products be analyzed on polyacrylamide gels, which have major constraints in high throughput screening (HTS) applications. Preparing the gels is a time-, labor-, and material-intensive task, especially at high volume.

FlashPlate is a 96-well polystyrene microplate with plastic scintillator coated wells, available exclusively from PerkinElmer Life Sciences. The platform is compatible with automated assay equipment such as the TopCount Microplate Scintillation and Luminescence Counter from Packard Instrument Company.

Materials

Streptavidin FlashPlate PLUS (SMP103)

Oligonucleotides

#1 (60 mer)
5' biotin-GGTTTAAAAA ATAGGAGGGA CAACGTCGTG
ACTGGGAAAA CTCCCCGGGT ACCGAGCTCG-3'

#2 (22 mer) 5'-GTTTTCCCAG TCACGACGTT GT-3'

SV40 Large T-Antigen (Molecular Biology Resources Inc., Milwaukee, WI)

Assay Buffer (50 mM Tris HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.5 mM MnCl2, 20% glycerol and 1
'"/>

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