Abstract
We developed a nicking endonuclease dependent DNA amplification (NDA), using Nt.BstNBI to catalyze single-stranded nick on double-stranded DNA, and Bst DNA polymerase to make extension while sealing the nick and displacing the downstream strand. The displaced single-stranded DNA thereby serves as template for primers hybridization and extension, resulting in exponential synthesis of target DNA under isothermal condition. Over 105 folds target DNA amplification can be achieved in 30 minutes, generating DNA product suitable for both diagnosis and DNA cloning. This NDA strategy does not require thermal cycling or prerequisite nucleotides modification, making it suitable for application in the field and at the point-of-care.
Highlights
DNA amplification is essential to most biological research involving nucleic acid manipulation
Using nickase instead of regular restrictive enzymes could greatly simplify the strategy of traditional strand displacement amplification
Successful reports about using nickase in amplifying target sequences are rare, if Figure 2. 2% agarose gel electrophoresis of 131 and 133 - bp nicking endonuclease dependent DNA amplification (NDA) products amplified from lambda DNA
Summary
DNA amplification is essential to most biological research involving nucleic acid manipulation. Several isothermal DNA amplification methods have been developed [3]. Strand displacement amplification (SDA) combines the ability of a regular restrictive endonuclease to nick a half-modified double-stranded DNA (dsDNA) and the action of an exonuclease-deficient DNA polymerase to extend the 3’ end at the nick while displacing the downstream strand [4,5,6]. Loop-mediated isothermal amplification (LAMP) employs a DNA polymerase and a set of four specific primers that recognize six distinct sequences on the target DNA, generating cauliflower-like stem-loop DNAs formed by annealing between inverted repeats [7]. Reported in 2004, helicasedependent amplification (HDA) uses a DNA helicase to separate dsDNA and generate single-stranded templates for primer hybridization and subsequent extension [8,9]. Most of the methods above need complex experimental procedures and their products are either too short to be used in further investigation or not compatible for cloning
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