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. 2009 Feb;37(2):e14.
doi: 10.1093/nar/gkn956. Epub 2008 Dec 10.

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

Michiko Kimoto  1 , Rie Kawai, Tsuneo Mitsui, Shigeyuki Yokoyama, Ichiro Hirao
Affiliations

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

Michiko Kimoto et al. Nucleic Acids Res. 2009 Feb.

Abstract

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3'-->5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10(7)-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.

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Figures

Figure 1.
Figure 1.
Structures of the unnatural Ds–Px and natural A–T and G–C pairs. R = aminohexanamide (for NH2-hx-Px) or (fluorescein-5-carboxamido)hexanamide group (for FAM-hx-Px). Space filling models of the base alone (with methyl in place of deoxyribose and R = H in the Px base) are shown, with electrostatic potentials mapped on van der Waals surfaces (PM3 calculations, Spartan ‘06, Wavefunction Inc.).
Figure 2.
Figure 2.
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 NNNDsNNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, dDsTP and FAM-hx-dPxTP, 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 NH2-hx-dPxTP, instead of FAM-hx-dPxTP (Figure 3a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM dPa′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.
Figure 3.
Figure 3.
PCR amplification of DNA fragments (55-mer) containing one Ds base. (a) Scheme for PCR amplification in the presence of FAM-hx-dPxTP or NH2-hx-dPxTP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx-Px, the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH2-hx-dPxTP and dDsTP 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 32P-labeled 5′-primer, in the presence of FAM-hx-dPxTP and dDsTP. 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 dPa′TP (2 μM) or ddPa′TP (50 μM). The arrows indicate the unnatural base position.
Figure 4.
Figure 4.
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-dPxTP or NH2-hx-dPxTP 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 32P-labeled 5′-primer, in the presence of FAM-hx-dPxTP and dDsTP. 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 32P-labeled 5′-primer, in the presence of NH2-hx-dPxTP and dDsTP. (d) Sequencing of the 15-cycle amplified DNA fragments, in the presence of dPa′TP (50 μM). The PCR products amplified in the presence of NH2-hx-dPxTP and dDsTP were used for sequencing of the Ds-strands. The arrows indicate the unnatural base position.
Figure 5.
Figure 5.
Detection of a target DNA molecule within foreign DNA fragments with the same primer sequences. (a) Overview of the purification of the DNA fragments containing Ds. After 15 cycles of PCR amplification of four different DNA fragments (55-mer, 0.15 nM each; one fragment contains one Ds base and the others contain no Ds bases) in the presence of FAM-hx-dPxTP and dDsTP, the amplified products were sequenced both directly and after purification using an anti-fluorescein antibody bound to magnetic beads. (b) Direct sequencing of the 15-cycle amplified DNA mixture, after removal of primers and dNTPs by gel purification, in the presence of dPa′TP (2 μM). (c) Sequencing of the DNA mixtures after purification of the FAM-labeled PCR products, in the presence of dPa′TP (2 μM).
Figure 6.
Figure 6.
Detection of a target DNA molecule within foreign DNA fragments with random sequences. Overview of the detection of the target DNA (DNA S2, 55-mer) containing Ds. A Ds-containing DNA fragment (DNA S2, 55-mer, 6 fM) and foreign DNA fragments with random sequences (100-mer, 60 nM) were amplified by 30 cycles of PCR in the presence of FAM-hx-dPxTP and dDsTP. After the amplification, the FAM-labeled PCR products were isolated by the magnetic bead-bound anti-fluorescein antibody, and then were sequenced in the presence of dPa′TP (2 μM). Prior to the antibody isolation, the amplified DNA fragments corresponding to the 55-mer were purified by gel electrophoresis and were also sequenced.
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