Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

Abstract

In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1 h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3-10 ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification.

Figure 1.

The bacteriophage T4 replisome. The bacteriophage T4 replisome is composed of eight proteins. The hexameric helicase (gp41) and oligomeric primase (gp61) constitute the primosome. Gp41 requires the helicase loader (gp59) for effective loading on the replication fork. Double-stranded DNA is unwound by the helicase. The single-stranded DNA thus generated is prevented from reannealing by binding of gp32. Gp61 synthesizes pentaribonucleotide primers (blue) that are then extended by the DNA polymerase (gp43). The polymerase, the clamp gp45 (trimer) and clamp loader proteins (gp44/62) form the holoenzyme. Gp45 increases the processivity of the polymerase and is loaded by gp44/62.

Figure 2.

Mechanism of cNDA. (A) One strand of the circular template is cut by the nicking endonuclase (1). The nick serves to initiate amplification by the T4 replisome (2). A new DNA strand is synthesized and single-stranded DNA is displaced (3). The nicking endonuclease also nicks the newly synthesized strand (4). The displaced strand is released from the template as it is cut at the nicking site (5). The ssDNA is primed by the reverse primer and the T4 replisome makes it double stranded (6). Linear copies of the circular template DNA result (7). (B) One single product of cNDA after amplification of the plasmid pIVEX-RFP.

Figure 3.

Restriction digests of cNDA product. (A) The plasmid pIVEX-RFP (4.3 kbp) was used as the template for amplification. The recognition site for the nicking endonuclease Nb.BbvCI used in cNDA is cut by the restriction enzyme BbvCI. If cut additionally with NdeI, two fragments with the lengths of 1.04 kbp and 3.26 kbp are generated. (B) Expected product of cNDA. (C) The product of cNDA (lane 1) was cut with BbvCI (lane 2), NdeI (lane 5) or BbvCI and NdeI (lane 6). The plasmid pIVEX-RFP (lane 8) was also digested with BbvCI (lane 3), NdeI (lane 4) or BbvCI and NdeI (lane 7).

Figure 4.

Real-time monitoring and amplification factors of cNDA. (A) Twenty microlitres of cNDA reactions containing 4 ng (red circles), 1 ng (green triangles), 0.25 ng (blue diamonds) and 0 ng (black squares) of template plasmid (pIVEX-GFP) were supplemented with the dsDNA intercalating EvaGreen dye. The reactions were carried out in a qPCR machine (Rotor-Gene 6000, Corbett) at 37°C and the fluorescence was recorded every 3 min for 6 h. (B) Twenty microlitres of cNDA reactions containing 8 ng (lane 1), 0.8 ng (lane 2), 80 pg (lane 3) and 8 pg (lane 4) of template plasmid (pIVEX-AGT-GST, 4.7 kbp) were incubated for 1 h. The products were run on an agarose gel (1%) and the amplification factors [shown at the bottom of (B)] were determined by qPCR.

Figure 5.

Amplification from crude E. coli lysates. cNDA was carried out on three colonies (each containing ∼60 000 cells) transformed with pIVEX-RFP. No band was detected from crude lysates without amplification (data not shown).

Figure 6.

cNDA followed by IVTT. The plasmid pIVEX-PTE [0.66 ng in (A) or 0.066 ng in (B)] was amplified by cNDA. Then, an IVTT mixture was added and PTE was expressed. Its activity was assayed by hydrolysis of the substrate paraoxon giving 4-nitrophenolate. The black progress curves (open squares) resulted from amplified DNA, whereas the red progress curves (closed circles) were from controls without DNA amplification.

Figure 7.

Immobilization of cNDA products. (A) cNDA reactions of pIVEX-PTE were performed with a non-biotinylated (b1, lane 1) and a biotinylated primer (biotin-b1, lane 2). (B) Reaction products from cNDAs with 10 ng or 1 ng pIVEX-PTE as template using primers b1 or biotin-b1 were immobilized on streptavidin-coated beads and added to IVTT mixtures. The activity of the expressed phosphotriesterase was assayed by hydrolysis of the substrate paraoxon. Red closed circles: 10 ng template DNA and biotin-b1 primer; green closed triangles: 1 ng template DNA and biotin-b1 primer; black open squares: 10 ng template DNA and untagged b1 primer.

Figure 8.

T4 pWGA product. Human genomic DNA (10 ng) was used as the template. The amplification yields products of different lengths.

Figure 9.

Real-time quantitative T4 pWGA. (A) Normalized real-time amplification signal of T4 pWGA containing 1 ng (red), 100 pg (blue), 10 pg (green), 1 pg (purple) and 0 (NTC; no template control; brown) human genomic DNA on a logarithmic scale. The threshold used for the quantification is drawn as a black line. (B) The time it took to cross the threshold (T_T) was plotted against the amount of input DNA. The blue circles are the samples containing input DNA and the red circles correspond to controls with no template. The linear fit (black line) corresponds to the following equation: T_T= –13.01 log₁₀[input DNA] + 37.47 (R²= 0.991).