Microsphere Polymerase Chain Reaction to Amplify Single-Stranded DNA

Abstract

Source: Lee, S. H. et al., A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons. J. Vis. Exp. (2018).

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

1. 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.
2. Gently vortex all reagents and then briefly centrifuge tubes at 10,000 x g for 10 s.
3. Combine all reagents as described in Table 1.
4. Place samples into a thermocycler and start the asymmetric PCR under the following conditions: 25 cycles (95 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s), 85 °C for 5 min, hold at 4
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Results

Figure 1: Illustration of the microsphere-PCR protocol.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Tris	Biosesang	T1016	Components of TE buffer, pH buffer solution
EDTA	Sigma Aldrich	EDS	Components of TE buffer, removal of ion (Ca2+)
Ex Taq	Takara	RR001A	ssDNA amplification
T100 Thermal Cycler	Bio-rad	1861096	ssDNA amplification