Helicase-dependent isothermal DNA amplification

Abstract

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

Figure 1

Schematic diagram of HDA. Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black triangle) separates the two complementary DNA strands, which are bound by SSB (grey circles). 2: Primers (lines with arrow heads) hybridize to the target region on the ssDNA template. 3: A DNA polymerase (squares with mosaic patterns) extends the primers hybridized on the template DNA. 4: Amplified products enter the next round of amplification.

Figure 2

Electrophoresis of HDA products amplified from plasmid DNA. A two-step HDA reaction, with a 1 h incubation at 37°C, was performed in the presence of all components (lane 1) including a pUC19-derived plasmid DNA (0.035 pmol), primer-1224 (10 pmol) and primer-1233 (10 pmol), UvrD helicase (100 ng), MutL (400 ng), T4 gene 32 protein (4.5 μg), ATP (0.15 μmol) and exo⁻ Klenow polymerase (5 U). HDA products in the absence of UvrD helicase (lane 2), accessory protein MutL (lane 3), T4 gene 32 protein (lane 4) or ATP (lane 5) are shown. M: 100-bp DNA ladder.

Figure 3

Electrophoresis of HDA products amplified from bacterial genomic DNA. (A) Amplification of a 123-bp target sequence from T. denticola genomic DNA. A two-step HDA reaction, with a 3 h incubation at 37°C, was performed in the presence of primer Ea136for (20 pmol) and primer Ea136rev (20 pmol), UvrD helicase (100 ng), MutL (800 ng), T4 gene 32 protein (4.5 μg), ATP (0.15 μmol) and exo⁻ Klenow polymerase (5 U). The copy number of the single T. denticola chromosome initially present in each HDA reaction is shown above each lane. (B) Amplification of a 102-bp target sequence with (lane 1) or without (lane 2) heat denaturation at 95°C, before a 2 h incubation at 37°C. The reaction was set up as described in (A), except that the primers used were Ea1for and Ea81rev. (C) Amplification of a 111-bp target sequence from T. denticola cells. A two-step HDA reaction, with a 10 min incubation at 95°C and 2 h incubation at 37°C, was performed. The reaction was set up as described in (A), except that the primers used were Gp98for and Gp188rev and 5.8 μg of RB49 gene 32 protein replaced 4.5 μg of T4 gene 32 protein. Frozen T. denticola cells were diluted in water and the initial amount present in each HDA reaction is shown above each lane. M: 100-bp DNA ladder.

Figure 4

Electrophoresis of 99-bp HDA products amplified from B. malayi genomic DNA in human blood samples. A 0.1–1,000 ng portion of B. malayi genomic DNA was added to 200 μl of human blood samples. After processing, 1 μl of each sample was used as template for HDA reactions. The amount of B. malayi genomic DNA initially present in each HDA reaction is shown above each lane. A two-step HDA reaction, with a 2 h incubation at 37°C, was performed. M: 100-bp DNA ladder.

Figure 5

Real-time HDA. A 97-bp fragment from T. denticola genomic DNA was amplified using a LUX primer. (A) Amplification products were detected in real time by measuring fluorescent signals (relative fluorescence unit (RFU)). Curves 1 and 2: two identical reactions performed on 10 ng of genomic DNA. Curve 3: reaction similar to curves 1 and 2 but without any genomic DNA (negative control). (B) Re-plotting data points corresponding to the early phase of the reaction, shown in (A), as the log of amplified products against time. Doubling time was calculated using the formula t_1/2=ln 2/V. (C) Electrophoresis of final amplified products. M: 100-bp DNA ladder.