Abstract
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. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products. Here we report the amplification of discrete target fragments of several kilobases at 37 °C from both double- and single-stranded circular template DNA using specific primer pairs. 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. 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. 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. 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.
Highlights
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
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
To assess the role of gp[32] in facilitating initiation, we tested it in DNA amplification reactions containing a pair of specific primers flanking an amplicon using circular double-stranded pUC19 plasmid template, DNA polymerase, dNTPs in a reaction buffer suitable for amplification (Fig. 1A)
Summary
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. 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. Primer design in methods like LAMP enable rapid exponential amplification as the primers form loop structures to create 3′ ends extendable by a DNA polymerase. All of these methods target very short amplicons, with length usually less than 150 base pairs (bp). We present a novel isothermal amplification scheme capable of producing discrete, several kilobase (kb)-long dsDNA products using only two specific primers, a strand-displacing DNA polymerase, and the single-stranded DNA binding protein gp[32] from bacteriophage T4. Gp[32] is used in the strand-invasion based isothermal amplification RPA6 and SIBA5 protocols where it works in concert with UvsX and UvsY to invade and anneal primers into dsDNA targets enabling exponential DNA amplification
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
