Figure 2

From: A general solution for opening double-stranded DNA for isothermal amplification

Figure 2

(A) 160 pmol RecA was incubated with 12 pmol 54 nt oligonucleotides in the presence of different nucleotide cofactor. Lane 1, no nucleotide. Lane 2, ATP. Lane 3, ADP. Lane 4, ATPγS. Lane 5, no RecA was added. (B) Incremental concentration of RecA was incubated with 12 pmol 54 nt oligonucleotides in the presence of ATPγS. Lane 1–6 correspond 20, 40, 80, 120, 160, 0 pmol RecA was added respectively. (C) Schematic of the process of strand exchange promoted by RecA. RecA and isotope-labeled ssDNA (ss) can form a nucleoprotein complex, then this complex will recognize its homologous double-stranded DNA (ds) and displace the identical strand to form a new isotope-labeled heteroduplex. RecA will disassemble from the product upon ATP hydrolysis and recycle. (D) Result of RecA promoted strand exchange between ssDNA oligonucleotides and homologous double-stranded DNA, reaction details could be found in Materials and Methods except the nucleotides cofactor variation: Lane 1, no nucleotides; Lane 2, 1 mM ATP; Lane 3, 1 mM ATP with 10 mM phosphocreatine and 1 U creatine kinase; Lane 4, 5 mM ATP; Lane 5, 5 mM dATP; Lane 6, 5 mM ATPγS; Lane 7, no RecA; Lane 8 and 9 are isotope-labeled ds and ss as markers.