Abstract
We herein describe a simple and versatile approach to use conventional nicking endonuclease (NEase) for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC), and its application to DNA isothermal exponential amplification (AMC-based strand displacement amplification, AMC-SDA). AMC uses a hairpin-shaped DNA aligner (DA) that contains a recognition site in its stem and two side arms complementary to target DNA. Thus, it enables the loading of an NEase on DA's stem, localization to a specific locus through hybridization of the side arms with target DNA, and cleavage thereof. By using just one NEase, it is easy to make a break at any specific locus and tune the cleavage site to the single-nucleotide scale. This capability also endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence. Moreover, this manner of initiation facilitates the adoption of 3'-terminated primers, thus making AMC-SDA highly sensitive and highly specific, as well as simple primer design.
Highlights
Ampli ed detection of nucleic acids with high sensitivity and selectivity is important in many elds such as molecular biology, medical diagnostics and forensic analysis.[1,2,3] At present, there are two main strategies for nucleic acid ampli cation: polymerase chain reaction (PCR) and isothermal ampli cation.[4]
By using two similar DNA aligner (DA), DA-1 and DA-2, which differ only in an extra A-T pair beyond the recognition site of DA-2 (Fig. 1a), a new, shorter band can only be found in the case of DA-2/T-1/ Nt.BstNBI, along with the intact DA-2 band and the disappearance of target DNA T-1 (Fig. 1a)
Two or more base pairs beyond the recognition site will lead to the digestion of DA itself, either with or without
Summary
Ampli ed detection of nucleic acids with high sensitivity and selectivity is important in many elds such as molecular biology, medical diagnostics and forensic analysis.[1,2,3] At present, there are two main strategies for nucleic acid ampli cation: polymerase chain reaction (PCR) and isothermal ampli cation.[4]. This capability endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence.
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