Linear Amplification
This step anneals biotinylated primer specific to the mobilized transposon and generates a linearly-extended single strand that spans the genomic junction of the transposon. Sequence of the biotinylated primer is specifically designed to hybridize with a unique sequence of the transposon reporter or transposition-mediated transgene, with the sufficiently long extension time to span the transposition breakpoint. In the case of this protocol designed to identify synthetic piggyBac transposon insertions, the extension time was determined by the 100 nucleotides separating the primer from the DNA transposon ITR (Fig. 1A).
- Prepare the PCR mixture in GeneMate UltraFlux tubes by mixing:
1 µL of 5 μM Biotinylated primer (100 nM)
4 μL genomic DNA (2 μg)
45 µL Invitrogen Platinum high fidelity SuperMix
- Carry out annealing and linear extension using the following PCR conditions:
95°C for 5 min
30 cycles of :
95 °C (45 seconds)
62 °C (45 seconds)
72 °C (3 min)
1 x 72 °C for 10 min
4 °C until next step
Transfer PCR reaction products into Amicon Ultra 0.5 100K filter tubes, add 200 μl of nuclease-free water
Centrifuge at 12,000 g for 10 minutes at room temperature, 42 μl concentrated purified sample should remain
Invert the column in clean tubes and centrifuge at 1,000 g for 2 min
Isolation of Biotinylated Amplicon
Transfer 3 μL of Dynal magnetic streptavidin beads into GeneMate UltraFlux tubes
Place the tubes onto DynaMag 96 magnetic holder
Remove supernatant
Add 20 µL kilobaseBINDER kit Binding solution
Place tube onto DynaMag 96
Remove solution
Resuspend magnetic beads in 10 μL Binding solution
Add 5 µL of isolated PCR product from the previous step
Add 5 μL of nuclease-free water
Shake tube at room temperature for 3 hours
Purification of Biotinylated Amplicon
Place tube onto DynaMag 96
Remove supernatant
Add 40µL of kilobaseBINDER kit Washing solution
Place tube onto DynaMag 96
Remove 40 μL Washing solution
Add 40 µL of nuclease-free water
Remove 40 μL water
Add 20 µL of 0.1M NaOH
Shake for 30 min at 37 C
Place tube onto DynaMag 96
Remove NaOH
Add 40 μL nuclease-free water
Place tube on DynaMag 96
Remove water
Synthesis of Complementary Strand
This step uses a degenerate primer to anneal and synthesize the complementary strand of the biotinylated amplicons. The sequence of the anchor primer was adapted from Pule et al. (1). The primer has a 3’ degenerate sequence and a 5’ adapter sequence that enables the anchor primer to prime to the 3’ end of the linear product of the previous step, containing the unknown genomic sequence flanking the inserted transposon (Fig. 1B).
Prepare the PCR buffer mixture by mixing:
15 µL nuclease-free water
2 µL 10x T7 DNA Polymerase Buffer
2 µL of 100 µM Anchor primer
1 µL of 10 mM dNTP (0.5 mM)
Add 19 µL of this mixture to tube with streptavidin beads and mix by pipetting
Incubate at 95°C for 1 minute and decrease temperature to 37 °C over 10 minutes
Centrifuge to collect any condensate and add 1 µL of T7 DNA polymerase
Incubate at 37 °C for 1 hour
At this point a linear double stranded DNA product has been synthesized, which spans the transposon terminal repeat as well as the flanking genomic sequence.
Product Amplification
This step uses exponential PCR to amplify the isolated double-stranded DNA sequences using adapter sequences included in the primers used for linear extension and complementary strand synthesis. As a result of this PCR, an amplicon is produced that includes sequences of the transposon terminal repeats and the flanking genomic sequences (Fig. 1C).
Wash the streptavidin beads to remove remaining anchor primers and carry out PCR:
Place tube on DynaMag 96 and remove supernatant
Add 40 μL of water
Place tube on DynaMag 96 and remove supernatant
Repeat 4 times
Prepare the PCR mixture:
1 µL of 25 µM Transposon1 primer
1 μL of 25 μM Exponential primer
45 µL of Invitrogen Platinum High Fidelity SuperMix
Add 48 μL of PCR mixture to the beads and mix by pipetting
Carry out PCR using the following conditions:
95 °C for 5 minutes
35 cycles of:
95 °C for 45seconds
62 °C for 45seconds
72°C for 3 minutes
1x 72 °C for 10 minutes
4 °C until next step
Nested Product Amplification
In order to increase the yield of specific amplicons spanning the breakpoint between transposon and flanking human genome, a nested PCR can be used (Fig. 1D).
Use PureLink PCR Purification Kit to purify PCR product amplicons. Elute in 50 µL nuclease-free water
Prepare PCR reaction mixture:
1 μl of 25 μM Transposon 2 primer
1 µl of 25μM Exponential primer
45 μl Invitrogen Platinum High Fidelity SuperMix
Add 3 µl of purified PCR product from previous PCR amplification to 47 μL of PCR mixture in GeneMate UltraFlux tubes
Carry out PCR using the following conditions:
95 °C for 5 minutes
35 cycles of:
95 °C for 45seconds
62 °C for 45seconds
72°C for 3 minutes
1x 72 °C for 10 minutes
4 °C until next step
- Purify PCR product and proceed with cloning for capillary Sanger DNA sequencing or library preparation for massively parallel single molecule DNA sequencing.