This video describes the technique of amplifying single-stranded DNA using oligonucleotide-coated polymer microspheres in a single-tube reaction. This process yields pure single-stranded DNA, which can be further used for DNA sequencing and microarray analysis.
Protocol
1. Asymmetric PCR for Amplifying ssDNA Prepare an asymmetric PCR reagent mix for amplifying ssDNA to be analyzed. Thaw the reagents in Table 1 on ice. Do not keep the Taq polymerase enzyme (50 U/µL) on ice, but rather store it at 20 °C until needed. Gently vortex all reagents and then briefly centrifuge tubes at 10,000 x g for 10 s. Combine all reagents as described in Table 1. Place samples into a thermocycler and start the asymmetric …
Representative Results
Figure 1: Illustration of the microsphere-PCR protocol.
Disclosures
The authors have nothing to disclose.
Materials
40% Acrylamide:bis solution (19:1)
Bio-rad
1610140
Components of Co-polymerizable oligo-microsphere
Ammonium persulfate, APS
Sigma Aldrich
A3678
Hardener of acrylamide:bis solution
N,N,N′,N′- Tetramethylethylenediamine, TEMED
Sigma Aldrich
T9281
Catalyst of ammonium persulfate
Mineral oil
Sigma Aldrich
M5904
Table 1. Solution III. Component of microsphere reagents
ssDNA acrydite-labeled probe
Bioneer
Synthesized
Table 1. Solution I. Component of microsphere reagents