Journal: Nucleic Acids Research

Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

doi: 10.1093/nar/gkn956

Figure Lengend Snippet: Overview of in vitro selection using a Ds -containing DNA library. ( a ) Scheme for in vitro selection using the Ds -containing DNA library, by FAM-hx- Px incorporation into PCR products and isolation with an anti-fluorescein antibody. A chemically synthesized, single-stranded DNA library containing an NNN Ds NNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, d Ds TP and FAM-hx-d Px TP, with DeepVent DNA Pol. After selection of the PCR products containing FAM-hx- Px by binding with an anti-fluorescein antibody, the isolated DNA fragments were used as a template for the next round of PCR amplification and selection. For direct sequencing of the library after five rounds of selection, the isolated DNA fragments were amplified in the presence of NH 2 -hx-d Px TP, instead of FAM-hx-d Px TP ( a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM d Pa′ TP, using a BigDye terminator v1.1 Cycle Sequencing kit. For the sequencing of clones after five rounds of selection, the library was amplified in the presence of only the natural dNTPs with Taq DNA polymerase, and the PCR products were used for TOPO TA cloning. ( b ) Sequencing of the DNA library after five rounds of selection. ( c ) Sequencing of the initial library. The arrow indicates the unnatural base position. ( d ) Probability (%) of occurrence at each position of the selected 66-clone sequences ( Supplementary Figure 1 ). Bases with an occurrence rate of ≥35% among the clones are colored red.

Article Snippet: PCR was carried out in a reaction buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 and 0.1% Triton X-100) with 2.5 μM FAM-hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP (N = A, G, C and T), 1 μM each of a 5′-primer (20-mer, 5′-GATAATACGACTCACTATAG-3′) and a 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.04 U/μl DeepVent DNA polymerase (NEB).

Techniques: In Vitro, Selection, Isolation, Synthesized, Sequencing, Amplification, Binding Assay, Clone Assay, TA Cloning

Journal: Nucleic Acids Research

Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

doi: 10.1093/nar/gkn956

Figure Lengend Snippet: PCR amplification of DNA fragments (55-mer) containing one Ds base. ( a ) Scheme for PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx- Px , the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds strands. ( b ) Polyacrylamide-gel analysis of the DNA fragments amplified by 15 or 30 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on a gel was detected with a bio-imaging analyzer, FLA-7000, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. The fold amplification of each DNA fragment is summarized in Table S3. ( c ) Sequencing of the 40-cycle amplified DNA fragments, in the presence of d Pa′ TP (2 μM) or dd Pa′ TP (50 μM). The arrows indicate the unnatural base position.

Article Snippet: PCR was carried out in a reaction buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 and 0.1% Triton X-100) with 2.5 μM FAM-hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP (N = A, G, C and T), 1 μM each of a 5′-primer (20-mer, 5′-GATAATACGACTCACTATAG-3′) and a 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.04 U/μl DeepVent DNA polymerase (NEB).

Techniques: Amplification, Sequencing, Labeling, Fluorescence, Imaging, Radioactivity, Autoradiography

Journal: Nucleic Acids Research

Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

doi: 10.1093/nar/gkn956

Figure Lengend Snippet: PCR amplification of DNA fragments containing two Ds bases (60-, 62-, 65- or 68-mer). ( a ) Scheme for the PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. The amplified DNA fragments containing FAM-hx- Px were detected by denaturing PAGE. ( b ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on the gel was detected with an FLA-7000 bio-imager, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. ( c ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of NH 2 -hx-d Px TP and d Ds TP. ( d ) Sequencing of the 15-cycle amplified DNA fragments, in the presence of d Pa ′TP (50 μM). The PCR products amplified in the presence of NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds -strands. The arrows indicate the unnatural base position.

Article Snippet: PCR was carried out in a reaction buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 and 0.1% Triton X-100) with 2.5 μM FAM-hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP (N = A, G, C and T), 1 μM each of a 5′-primer (20-mer, 5′-GATAATACGACTCACTATAG-3′) and a 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.04 U/μl DeepVent DNA polymerase (NEB).

Techniques: Amplification, Labeling, Fluorescence, Radioactivity, Autoradiography, Sequencing