Microsphere Polymerase Chain Reaction to Amplify Single-Stranded DNA

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 °C.

2. Microsphere-PCR for Amplifying ssDNA

NOTE: This section describes the protocol for amplifying ssDNA in a PCR reaction tube. Microsphere-PCR reactions were performed in 50 μL of reaction volume. The detailed sequences used to amplify ssDNA are listed in Table 2. In this case, Ap on the surface of microspheres can anneal to random DNA templates in a complementary manner. This is a very important step for producing complementary DNA strands (antisense DNA strand, Figure 1). The DNA extended is used as a template for microsphere-PCR amplification.

1. Prepare microsphere-PCR reagent mix. Thaw the reagents in Table 3 on ice; however, do not keep the Taq polymerase enzyme on ice. 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. Obtain approximately ~25 microspheres through microscopic counting.
   NOTE: The number of microspheres in one reaction tube is calculated using a light microscope with 40X magnification. About ~25 microspheres are used for microsphere-PCR amplification. More detailed information is in Step 3.
4. Combine all reagents as described in Table 3.
5. 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 °C.
6. Following amplification, add 8 μL of 6x loading buffer and load 15 μL of each sample into 2% agarose gel. Then, perform electrophoresis at 100 V for 35 min in 1x TAE (Tris-acetate-EDTA, 40 mM Tris-acetate, 1 mM EDTA, pH 8.2) buffer.

Table 1. Asymmetric PCR reagent mix components in 20 μL reaction.

Reagent	Volume for 1x reaction (μL)	Final concentration
Random DNA template	1	1 ng/μL
Tag-forward primer	1	0.4 μM
Reverse primer	1	0.02 μM
10X Taq buffer	5	1X
10 mM of dNTP	4	2.5 mM
Ex taq (1000U)	0.2	1 U
Water	37.8	-
Total	50	-

Table 2. Sequence information for Microsphere-PCR.

Template	Sequence	Length (nt)
Random DNA template	5'- ATA CCA GCT TAT TCA ATT (Random sequence, 40 mer) AGA TAG TAA GTG CAA TCT-3'	76
Tag-Forward primer (Tag-F)	5'- GGT AAT ACG ACT CAC TAT AGG GAG ATA CCA GCT TAT TCA ATT-3'	42
Reverse primer	5'- AGA TTG CAC TTA CTA TCT-3'	18

Table 3. Microsphere-PCR reagent mix components in 50 μL reaction

Reagent	Volume for 1x reaction (μL)	Final concentration
Ap-Microspheres	~25 microspheres	-
Random DNA template	1	1 ng/μL
Tag-Forward primer	1	0.4 μM
10X Taq buffer	5	1X
10 mM dNTP	4	2.5 mM
Ex taq (1000U)	0.2	1 U
Water	37.8	-
Total	50	-

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Results

Figure 1: Illustration of the microsphere-PCR protocol.

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Materials

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