Overview
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 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.
- 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.
- Gently vortex all reagents and then briefly centrifuge tubes at 10,000 x g for 10 s.
- 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. - Combine all reagents as described in Table 3.
- 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.
- 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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Representative 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 |
