Strand displacement amplification—an isothermal,in vitroDNA amplification technique

Abstract

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

Loop-mediated isothermal amplification of DNA

“…In addition to the widely used PCR-based detection (1,2), several amplification methods have been invented. They include nucleic acid sequence-based amplification (NASBA) (3), self-sustained sequence replication (3SR) (4) and strand displacement amplification (SDA) (5,6). Each of these amplification methods has its own innovation to re-initiate new rounds of DNA synthesis.…”

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

“…Strand-displacement reactions are known to permit DNA amplification in very high yields (Walker et al 1992;Lizardi et al 1998;Dean et al 2001). We have adapted the use of the strand-displacing polymerases of phage 29 and Bacillus stearothermophilus (Bst DNA polymerase large fragment, 5Ј → 3Ј exo ‫מ‬ ) for random-primed amplification of human genomic DNA.…”

Molecular Diagnosis of Enteric Fever: Progress and Perspectives

“…However, new PCR technologies, such as isothermal PCR, are of particularly practical use in the diagnosis of enteric fever, as these methods allow for the possibility of developing less-complicated and less-expensive machinery than is necessary for conventional PCR. Several isothermal PCR technologies have been developed (Gill & Ghaemi, 2008), including strand displacement amplification (SDA) (Walker et al, 1992), loop-mediated amplification (LAMP) (Notomi et al, 2000), and helicase-dependent amplification (HDA) (Vincent et al, 2004). Recently, Francois et al have examined the robustness of LAMP for bacterial diagnostic applications using S. Typhi as the target organism (Francois et al, 2011), and demonstrated that LAMP is more sensitive than conventional qPCR and is also a very robust, innovative and powerful molecular diagnostic method.…”