What lab techniques exist to differentiate between single-strand and double-stranded DNA?

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    – tyersome
    Commented Nov 2, 2019 at 23:08

1 Answer 1


Single and double stranded DNA have different electrophoretic mobilities, ssDNA will move faster on an agarose gel, so if you know the length of your DNA and it moves further than you expect relative to a dsDNA ladder it's probably ssDNA. The hyperchromic effect means that ssDNA also absorbs more strongly than dsDNA, so you can heat up your DNA and if the absorbance changes it was dsDNA and if it doesnt then its ssDNA (you should take an aliquot for this so that you have the rest of your sample in tact). You could also take an aliquot and add a single stranded exonuclease and run the products on a gel together with the DNA before the digestion, if nothing is present after in the digested well then you had ssDNA if there is still a band after then you had dsDNA (i don't think this method is really used as you could just run it on a gel which is simpler and you lose less sample, but it should still work).

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    $\begingroup$ +1 but I'd like to add an important caveat: intercalating dyes that allow visualization of dsDNA on a gel may not bind strongly or at all to ssDNA. So if OP does plan on doing gel visualization of mixed single- and double-stranded nucleic acids, consider using a dye like ethidium homodimer-1 or -2. Good source for dye selection here. $\endgroup$
    – acvill
    Commented Nov 4, 2019 at 15:13
  • $\begingroup$ yes thats true you do definitely see a weaker band, so you do have to load more DNA or use a more sensitive dye. I've done with a couple of 100 ng with sybr safe and that has been fine, but it depends if you have that to spare. $\endgroup$ Commented Nov 4, 2019 at 16:11
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    $\begingroup$ @Dirigible Actually the fact that certain dyes bind ssDNA & dsDNA with different strenght can itself be used to differentiate between them. (As long as concentrations are known / comparable). Many qPCR systems also rely on that to measure melting curves, i.e. using dyes like SYBR $\endgroup$
    – Nicolai
    Commented Nov 6, 2019 at 15:55
  • $\begingroup$ @pepsiandsoda I have a doubt...Wouldn't the absorbance also change for ssDNA as we heat it because it is also in some weird coiled shape and that shape must be due to hydrogen bonds which might not let the ssDNA to absorb the light fully. With increase in temperature, these bonds will break and absorbance must increase, although the increase might be small... $\endgroup$ Commented Feb 19, 2020 at 5:45

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