Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method - PubMed (original) (raw)

Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method

D Hern Paik et al. Nucleic Acids Res. 2013 Oct.

Abstract

Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two 'megaprimers', 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.

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Figures

Figure 1.

Figure 1.

Ligation-based assembly of DNA constructs for torsionally constrained assays. (A) Schematic of a single-molecule optical-trapping assay where the DNA is multiply labelled in both strands at both ends. Different tags [biotin (orange) and digoxigenin (cyan)] are incorporated into short DNA molecules (purple) that were ligated onto a central unlabelled DNA molecule. (B) An outline of the traditional method for making torsionally constrained DNA. In this method, three different DNA molecules are made by PCR, two of which are labelled with dig and biotin, respectively (Step 1). Restriction digest (Step 2) of all three DNAs facilitates the ligation necessary to assemble the final product (Step 3). Gel purification is necessary to achieve a uniform length product (Step 4).

Figure 2.

Figure 2.

PCR-based assembly of DNA constructs for torsionally constrained assays. (A) Schematic of a single-molecule optical-trapping assay, where the DNA is multiply labelled on one strand at each end. Different tags [biotin (orange) and digoxigenin (cyan)] are incorporated into the DNA megaprimers (purple, 400-bp) that were used to amplify the target DNA during PCR. (B) An outline of our ligation-free method for making torsionally constrained DNA. Briefly, we linearized the template (Step 1) to suppress the amplification of off-target products. In Step 2, two sets of ∼25-bp ssDNA primers are used in PCR to make the two labelled 400-bp dsDNAs that becomes the megaprimers for the subsequent PCR (Step 3). Finally, the target DNA is gel-purified away from the 400-bp dsDNA megaprimers (Step 4).

Figure 3.

Figure 3.

Analysis of PCR products and purification technique using non-denaturating gel electrophoresis. (A) An agarose gel showing intermediate and final products. Lane M: DNA ladder (1 kb+, Invitrogen); Lane 1: Biotin-labelled megaprimer; Lane 2: Dig-labelled megaprimer; Lane 3: Pre-gel purified megaprimer PCR showing the desired product and unincorporated megaprimers; Lane 4: Final PCR product (6592 bp) after gel purification. (B) Fractions from continuous elution electrophoresis. Lane M: DNA ladder (1 kb, NEB); Lane 1: Mixture of PCR products (before separation); Lanes 2–5: Fraction 2, 3, 10, 15 (unreacted megaprimers); Lanes 6–10: Fraction 21, 22, 23, 25, 30 (6592-bp DNA). Fractions 21–30 were combined. (C) Gel analysis of the pooled product from the continuous elution electrophoresis. Lane M: DNA ladder (1 kb, NEB); Lane 1: Purified and pooled DNA.

Figure 4.

Figure 4.

Torsionally constrained DNA probed by measuring the overstretching force. (A) Representative force-extension curves of torsionally constrained (top) and unconstrained (bottom) DNA that exhibited overstretching forces at 110 and 65 pN, respectively. The molecules were first stretched (light colour) and then relaxed (dark colour), with hysteresis in the lower panel indicative of peeling from a nick or free end (16). (Inset) Cartoon of a torsionally constrained and an unconstrained DNA anchored to a surface via multiple bonds at each end. The bond colour indicates biotin (orange) and digoxigenin (cyan), respectively. (B) Pie charts showing the percentage of torsionally constrained DNA at three independent conditions: (top) DNA made with high-density megaprimers and purified with the continuous-elution method; (middle) DNA made with high-density megaprimers and purified by standard gel purification including UV irradiation; and (bottom) DNA made with standard density megaprimers and purified by standard gel purification.

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