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Strategies to facilitate initiation provide the main variance among isothermal methods. coli DNA Polymerase I, or phi29 DNA polymerase) separates dsDNA after initiation at a primer, with the initiation step proving the key limit to speed and efficiency of an isothermal reaction. Approaches vary, but generally strand displacement activity by a DNA polymerase (typically the large fragment of Bsu, Bst, and E. Additionally, isothermal methods can provide extremely rapid results, as fast as 10 minutes, and can produce a large amount of DNA, enabling simple, visual detection of amplification 1, 2.Įach isothermal technique relies on enzymatic activities or primer design to bypass the need for thermal denaturation of dsDNA. These methods require simpler instrumentation, using only a single, moderate temperature. Recent years have seen growing adoption of methods that amplify DNA or RNA at a single temperature, accordingly referred to as “isothermal” amplification. Additionally, the degree of amplification is intrinsically limited by the cycling of the reaction: each cycle can at most produce a doubling of the DNA. While routine and flexible, PCR is limited in application by its requirement for instruments capable of thermal control and cycling between high temperatures for dsDNA denaturation and lower temperatures for primer extension by the DNA polymerase. DNA amplification can be performed using a variety of strategies, most commonly the polymerase chain reaction (PCR) which is enabled by cycles of thermal denaturation of double-stranded DNA (dsDNA).

Most methods involving DNA or RNA start with the molecules of interest at concentrations far below the necessary amount, and accordingly must be increased through amplification. Much of modern biotechnology and molecular biology relies on the ability to manipulate a specific nucleic acid target, but in order to efficiently detect, read, or use that target sequence, it must be available at sufficient quantities in the application. The ability presented here to produce long, discrete DNA products in an isothermal reaction extends the scope of isothermal amplification to enable more useful applications of these promising methods.

We demonstrate that two features of gp32 enable this amplification: a facilitation of primer strand invasion into double-stranded DNA, and a suppression of non-homologous primer annealing and nonspecific amplification.

In addition to the discrete amplicon products, this method also produces higher molecular weight products consisting of multiple repeated copies of the amplicon and template DNA. In contrast to existing methods, this amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. Here we report the amplification of discrete target fragments of several kilobases at 37 ☌ from both double- and single-stranded circular template DNA using specific primer pairs. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products.

Isothermal amplification methods for detection of DNA and RNA targets have expanded significantly in recent years, promising a new wave of simple and rapid molecular diagnostics.