Abstract
Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it.
Highlights
Exchange between dsDNA and single stranded primer to form D-loop
recombinase of E. coli (RecA) disassemble from ssDNA when ATP was added, which could be caused by ADP produced from hydrolysis of ATP as RecA/ssDNA has the strong ATPase activity
This assumption was verified by adding ADP into the reaction, where we found that RecA disassemble from ssDNA when ADP was added
Summary
Exchange between dsDNA and single stranded primer to form D-loop. the primer can be extended by DNA polymerase with strand-displacement activity (Fig. 1B). The substantial function of RecA in vivo is to form nucleoprotein complex with transient single-stranded DNA and facilitate strand exchange with homologous duplex[24]. We ask this question: Can we utilize the recombinase RecA of E. coli as a tool to open double-stranded DNA for isothermal detection methods. To achieve this goal, there are three challenges we need to address: 1. We report the research to figure out these problems and establish a general solution to open double-stranded DNA for the different isothermal detection methods using RecA protein from E. coli
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