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PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

Hommelsheim, Carl Maximilian, Frantzeskakis, Lamprinos, Huang, Mengmeng, Ülker, Bekir·2014·Scientific Reports
DOI 10.1038/srep05052PMC PMC4031481
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Figure 1 PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3. Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material.
Figure 1 PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3. Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 2 Cloning and sequencing of PCR artifacts. (A) The TALE repeats were amplified from the pTAL2 vector using primers 534 and 535. The indicated size of PCR amplification fragments were gel-isolated and cloned into pTOPO® vectors from Life Technologies. These clones were sequenced using M13 reverse and forward primers. (B) The alignments of sequencing results to the expected TALE repeat organization are shown graphically. E: expected residue organization and truncated versions. The sizes of the sequence fragments are indicated on the graphical display. Corresponsing sequences are given in the supplementary material.
Figure 2 Cloning and sequencing of PCR artifacts. (A) The TALE repeats were amplified from the pTAL2 vector using primers 534 and 535. The indicated size of PCR amplification fragments were gel-isolated and cloned into pTOPO® vectors from Life Technologies. These clones were sequenced using M13 reverse and forward primers. (B) The alignments of sequencing results to the expected TALE repeat organization are shown graphically. E: expected residue organization and truncated versions. The sizes of the sequence fragments are indicated on the graphical display. Corresponsing sequences are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 3 Proposed model explaining how the PCR artifacts are generated. Polymerization function is hindered by out of register template strand or PCR product alignments ahead of the polymerase. These partially amplified fragments act as mega primers in the following cycles and could bind to the template or other impartially generated fragments in or out of register, leading to the observed laddering effect.
Figure 3 Proposed model explaining how the PCR artifacts are generated. Polymerization function is hindered by out of register template strand or PCR product alignments ahead of the polymerase. These partially amplified fragments act as mega primers in the following cycles and could bind to the template or other impartially generated fragments in or out of register, leading to the observed laddering effect.CC BY-NC-SA · PubMed Central
Figure 4 Extension of proposed model explaining how the PCR fragments of discrete sizes, smaller and larger than the expected fragments, are generated.
Figure 4 Extension of proposed model explaining how the PCR fragments of discrete sizes, smaller and larger than the expected fragments, are generated.CC BY-NC-SA · PubMed Central
Figure 5 Testing of various polymerases under supplier's recommended conditions in the amplification of TALE DNA-binding repeats from the pTAL2 vector. The arrows indicate the expected 1551 bp size of the amplification product. PCR conditions are given in the supplementary material.
Figure 5 Testing of various polymerases under supplier's recommended conditions in the amplification of TALE DNA-binding repeats from the pTAL2 vector. The arrows indicate the expected 1551 bp size of the amplification product. PCR conditions are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 6 Testing a DNA polymerase with high strand displacement activity in the amplification of the 12 TALE DNA-binding repeats in pTAL2 vector. Various parameters influencing the activity and the outcome of the PCR amplification of the Deep-VentR DNA polymerase are shown. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material.
Figure 6 Testing a DNA polymerase with high strand displacement activity in the amplification of the 12 TALE DNA-binding repeats in pTAL2 vector. Various parameters influencing the activity and the outcome of the PCR amplification of the Deep-VentR DNA polymerase are shown. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 7 ET SSB slightly improves the performance of Deep-VentR DNA polymerase in amplifying TALE DNA-binding repeats. The arrow indicates the expected size of amplification products. ET SSB, a heat resistant single strand DNA-binding protein isolated from thermophilic bacteria (NE Biolabs). PCR conditions are given in the supplementary material.
Figure 7 ET SSB slightly improves the performance of Deep-VentR DNA polymerase in amplifying TALE DNA-binding repeats. The arrow indicates the expected size of amplification products. ET SSB, a heat resistant single strand DNA-binding protein isolated from thermophilic bacteria (NE Biolabs). PCR conditions are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 8 Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material.
Figure 8 Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 9 Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material.
Figure 9 Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material.CC BY-NC-SA · PubMed Central
Figure 10 Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.
Figure 10 Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.CC BY-NC-SA · PubMed Central
Figure 11 The model explaining how GFP sequences arranged as direct repeats separated by a linker DNA generate artifact single copy GFP. See supplementary Fig. 5 for the model describing the generation of artifacts in inverted repeats.
Figure 11 The model explaining how GFP sequences arranged as direct repeats separated by a linker DNA generate artifact single copy GFP. See supplementary Fig. 5 for the model describing the generation of artifacts in inverted repeats.CC BY-NC-SA · PubMed Central
Figure 12 Testing the generality of the model to other template DNAs with repetitive sequences and better-performing polymerases. See the supplementary material for PCR conditions.
Figure 12 Testing the generality of the model to other template DNAs with repetitive sequences and better-performing polymerases. See the supplementary material for PCR conditions.CC BY-NC-SA · PubMed Central
Figure 13 Increasing the distance of the primer annealing site to the repetitive GFP reduces artifacts. Notice that even for the primer combinations 572 + 390, the amount of artifact products were reduced and the amount of the correctly sized fragment was increased when the extension time increased from 2 minutes to 5 minutes (compared to Fig. 10). See supplementary Fig. 4 for the plasmid maps used in PCR amplification.
Figure 13 Increasing the distance of the primer annealing site to the repetitive GFP reduces artifacts. Notice that even for the primer combinations 572 + 390, the amount of artifact products were reduced and the amount of the correctly sized fragment was increased when the extension time increased from 2 minutes to 5 minutes (compared to Fig. 10). See supplementary Fig. 4 for the plasmid maps used in PCR amplification.CC BY-NC-SA · PubMed Central
Figure 14 Template switching by DNA polymerase might also be involved in the generation of PCR artifacts.
Figure 14 Template switching by DNA polymerase might also be involved in the generation of PCR artifacts.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Hommelsheim, C. M., Frantzeskakis, L., Huang, M., & Ülker, B.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2014-05-23
Volume
4
Issue
1
ISSN
2045-2322
DOI
10.1038/srep05052
PMC
PMC4031481
Cited by
121
References
2
Language
en
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