Study On The Nickase-dependent DNA Strand Displacement Amplification

Background and Object:In recent years, rapid development of isothermal nucleic acid amplification technology shows the potential application. Strand-displacement amplification is a sensitive, rapid and specific isothermal DNA amplification method. But presence of dNTP[αS] in the reaction limits the application of SDA in molecular biology. Nickase is a special kind of restrictive endonucleases that can catalyze single-stranded nick on natural double-stranded DNA without any modified nucleotides. Our study aims to combine nickase with Bst DNA polymerase to establish a simpler and more efficient DNA amplification method---Nickase Dependent Strand Displacement Amplification, which should be applied to detect some deseases and pathogens in primary health units.Methods:1) Amplification of short target sequence from Lambda DNA, to construct suitable system of amplification, to search for reaction condition, such as relative amount of nickase and polymerase, primers and templates, temperature, time, inonic strength, pH, and so on.2) To explore the possibility of amplification of longer target sequence(>500 bp) on the basis of above works.3) Amplification of specific sequences from human genome DNA, Neisseria gonorrhoeae genome DNA and Candida albicans genome DNA.Rusults:1) Have amplified 131/133 bp target sequence from Lambda DNA, have constucted the most suitable system of amplification of short target sequence by optimize the reaction condition. 2) Have amplified 200 bp, 500 bp target sequence from Lambda DNA.3) Have amplified 500 bp target sequence from pUC18 plasmid DNA.4) Have amplified 1.2 kb target sequence from Lambda DNA, but the production and purity need to be further improved.5) Have not amplified specific sequence from human genome DNA, Neisseria gonorrhoeae genome DNA and Candida albicans genome DNA.Conclusions:1) The reseach combines nickase with strand displacement amplification to establish initially a method, which is nickase dependent DNA strand displacement amplification (NDA) that can be used to amplify short target sequence from Lambda DNA and pUC18 DNA successfully. It is sensitive, rapid and specific.2) The suitable amplification condition of NDA containing 0.4μM dNTP, 1μM primers, 100 mM NaCl, 8U Bst DNA polymerase Large Fragment, 2U Nt.BstNBI in 25μL reactions, and reaction temperature is 55℃, reaction time is 15-30 minutes.3) NDA has potensial to amplify longer target sequence, for which the reaction system need to be further adjusted.