Isothermal DNA amplification in vitro: the helicase-dependent amplification system

Abstract

Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification.

Fig. 1

Schematic diagram of the Helicase-dependent DNA amplification. Helicases unwind dsDNA and SSB proteins bind to exposed ssDNA. Subsequently, DNA polymerases start synthesizing the complementary strand from the bound primers, and the cycles repeat continuously

Fig. 2

Model for the mechanism of UvrD loading on ssDNA. A model for loading onto ssDNA and dsDNA unwinding by UvrD + MutL at 37°C (a) or by thermostable UvrD alone at high temperature (60–65°C) (b). See text for details

Fig. 3

Model of T7 bacteriophage replisome at the replication fork. The schematic shows the DNA replication system of T7 bacteriophage, which consist of the DNA polymerase (gp5), the helicase-primase (gp4), the ssDNA-binding protein (gp2.5), and the processivity factor E. coli thioredoxin (trx). Hexameric helicase T7 gp4 unwinds the dsDNA at the fork and the primase domain of T7 gp4 produces the RNA primer (red segment). Extruded ssDNA (lagging strand) is coated by gp2.5 and aligns its polarity with the leading strand through a loop formation. The leading strand is synthesized by the T7 gp5/trx complex tethered to gp4 as Okazaki fragments, whereas the leading strand is synthesized by T7 gp5/trx complex continuously

Fig. 4

Schematic of circular helicase-dependent DNA amplification. The T7 replisome machinery consists of a T7 gp4B helicase and T7 DNA polymerase gp5/trx complex. Primer extension and strand displacement produces a concatamer of the circular template DNA. Multiple reverse primers anneal to the concatamer and are extended by the T7 DNA polymerase. The helicase/DNA polymerase complex displaces the non-template strands, which provide complementary sites for forward primers to anneal. Duplex DNAs are produced by the T7 replisome after the release of ssDNAs for the next round of strand displacement synthesis. This figure is reproduced and modified from [81]

Fig. 5

Mechanism of primase-based whole genome amplification. The T7 helicase-primase gp4 denatures the dsDNA template and synthesizes primers. Primers are extended by the T7 DNA polymerase gp5/trx complex, resulting in DNA replication in both strands. Newly synthesized DNA is displaced and serves as a template for whole genome amplification. This figure is reproduced and modified from [105]