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I've started reading about the recombinase polymerase amplification (RPA). I'm learning that in RPA, recombinase enzyme binds to primers, then makes them anneal to the complementary target DNA strand, and the displaced strand of DNA is bound by single-strand binding protein (SSBP) to prevent reannealing and promote extension of the bound primer. But how come the primers aren't bound by SSBP thus preventing them being bound by the recombinase? How come it's not recombinase that binds the displaced DNA strand? Materials that I'm reading don't mention anything about adding some kind of recombinase-recognised tags to the primer sequences, how do these two enzymes "know" which single stranded DNA to bind to?

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From a 1994 review of E.coli single-stranded binding protein (SSBP) in Annual Reviews of Biochemistry, it would appear that at least 35 nucleotides (nt) of single-stranded DNA are required for binding of SSBP to occur.

From a paper in Trends in Analytical Chemistry describing recombinase polymerase amplification I read that the first step in the procedure (A, below) is to add the primers to the recombinase, forming a complex between the two, and that it is only at a subsequent stage (D) that SSBP is added.

RPA

As the length of primers normally used is also apparently ca. 35 nt, I presume that the length of primer available to the SSBP is less than 35 nt, and it is for this reason that SSBP is unable to bind to it. (In the cartoon there would appear to be no primer available, except, presumably, for the DNA.)

An alternative would be that the recombinase hinders the binding of the SSBP.

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  • $\begingroup$ Thanks for the reply. Yes, I read that article, but it doesn't actually say that the SSBPs are added at a later stage. They only show up in panel D of the figure because that's where they're relevant, not because that's where they're added. Like the DNA polymerase is not added after the SSBP (panel E). Also, the manuals for commercial RPA kits do not specify that SSBPs are added after the primers, recombinase and template. However, your "less than 35nt" theory does make sense! And thanks for the edits to my question, but they weren't necessary. $\endgroup$ Nov 22, 2023 at 10:39
  • $\begingroup$ @AndrewRoots — If you found part of my answer helpful you should consider upvoting or accepting it. As regards your opinion that my edits weren't necessary, I provide an explanation of the reasons for such edits on Meta. It is, of course, your prerogative to disregard them, and I do not wish to discuss this matter further. $\endgroup$
    – David
    Nov 22, 2023 at 21:17

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