Biocurious is a weblog about biology, quantified.

Labeling lambda DNA

by PhilipJ on 14 January 2006

Whenever my lab gets a visitor (usually a physicist), after giving them the tour and showing off the optical tweezers instrument, I always get asked the same question: “so how do you attach DNA to beads, anyway?” We physicists are a clever bunch, but (bio)chemistry isn’t often in our bag of tricks. Since many of our readers are physicists too, here’s how we end-label DNA for force-extension measurements, using λ DNA as our DNA of choice, partially because you can buy it commercially, and also because its the DNA I’ve been labeling all day across the hall in our wet lab (using [1])!

A bit of background information first, unrelated to labeling: λ DNA is so-named because it is the genome for the λ phage, a virus that attacks E. coli. Shown on the right, it is particularly sinister-looking creature! The capsid (the round bulb at the top) holds and protects the DNA until it is ready to get injected into a bacterium. Once injected, the virus integrates into the host’s genome, tagging along into each new bacteria upon cell division and causing virtually no harm (called the prophage pathway). When the time is right (for example, when the host is signaling that it will die), the virus exits the host’s genome and uses the host’s replication machinery to create new copies of itself (the lytic pathway). Once all resources inside the cell are depleted, the cell lyses, freeing the λ phages to infect others.

Back to labeling! There are a couple of pairs of molecules that are commonly used when trying to make molecular handles, and I’ve mentioned the ones we use before, namely the antibody/antigen pair of digoxigenin and anti-digoxigenin, and streptavidin/biotin. They are so common that you can buy anti-dig and streptavidin (and many other) coated beads! Now we just need to find a way to attach biotins and digoxigenins to either end of our DNA. It turns out this isn’t as tricky as a physicist might imagine either — the demand is large enough that you can buy biotinylated and digoxigenined dNTPs! Now, lets see how we add them to our λ DNA.

Prior to injection in an E. coli host, λ DNA is a linear, double-stranded molecule with overhanging sticky (that is, having unpaired bases) ends, of total length 48,502 bases. It is in this form that you can buy it, and at fairly high concentrations too. Let’s take a closer look at the overhanging sticky ends:

Both sides have all four bases overhanging, but the order isn’t the same on either side! You’ll also note that there is a single free A on either side. If we are clever, we can do each side individually, and end up with a dig-dUTP on one end, and bio-dUTP on the other (U, uracil, is another amino acidbase (thanks Neil!) that will bind with A, adenosine). If we put λ DNA in a test tube with dATP, dGTP, bio-dUTP and a DNA polymerase (experts will know that we often use klenow exo-), the DNA polymerase will start encorporating the complementary bases on either end up until we need a C, cysteincytosine (note to self: don’t do this at so late anymore, thanks UndergradChemist!), which is currently being withheld. Letting that reaction occur, we get DNA which looks like:

Alright, halfway there! At this point we need to get rid of any excess bio-dUTP bases, and so one normally ethanol precipitates the DNA and resuspends the DNA in a buffer free of nucleotides. We’re now free to label the other end, so adding dATP, dGTP, dCTP, and dig-dUTP along with a DNA polymerase will fill in the rest. We end up with:

This is exactly what we need to now attach the DNA and start pulling. The way I’ve been doing the attachments is to incubate DNA labeled on either end with one of the sets of beads so that they are covered in many (up to ~1000) DNA molecules each, and “fishing” for the other tether using the micropipettes and the other complementary beads, eventually making a tether between the two like so:

So, there you have it! This same technique can be used to label more or less all DNA of interest, and in some situations it is an even easier single-step process (for example, when one end has only As and Ts overhanging, while the other has only Gs and Cs). Now, if only my yields weren’t so poor after the ethanol precipitation step…..

[1] R. M. Zimmermann & E. C. Cox, DNA stretching on functionalized gold surfaces, Nucleic Acids Research 22 492-497 (1994).



  1. UndergradChemist    4088 days ago    #
    I’m just curious, is there any particular problem involved with measuring forces and tension if the fixtures at the ends of the DNA aren’t covalent? Do you have to compensate for that in calculations, or are the bonds strong enough that the differences aren’t significant?

    (By the way, I think you meant cytosine for C)
  2. Neil    4088 days ago    #
    You do mean cytosine for C and you mean “base” for uracil, not “amino acid”.
  3. Andre    4087 days ago    #
    Hi UndergradChemist,

    I don’t know about optical tweezers, but certainly when AFM is used for pulling experiments covalent attachment is not necessary. The vast majority of extension experiments (excluding ligand/receptor studies) are done with molecules physisorbed on a surface. The AFM tip is then touched on the surface and sometimes something attaches. When it does, you can pull it and actually achieve forces as high as several hundred pico newtons before the molecule finally desorbs. This force is around the upper range that optical tweezers can achieve, so I don’t think the strength of the attachment is the reason optical tweezers experiments rely on specific attachment.

    I will speculate that it’s because the radius of curvature of the beads used in optical trap experiments is much larger than the radius of curvature of a sharpened AFM tip (~20nm). That means that a bead is unlikely to contact just a part of a molecule like DNA to allow pulling, instead it will likely just mash the molecule and then unbind it from the surface giving you some information about how strongly it sticks to beads, but nothing about its mechanics. But again, I’m just speculating…

    Another advantage of specific attachment is that it simplifies the interpretation since you know the geometry of your experiment. One of the hardest parts in an AFM pulling experiment is interpreting your data because the attachment is random and throughout the course of your experiment you will have probably sampled all along the length of your molecule. That’s why force spectroscopy with AFM is typically done with long chains of the same protein domain repeated several times. It allows you to actually figure out what’s going on!
  4. PhilipJ    4087 days ago    #
    As you can tell, even remembering simple names of things in (bio)chemistry doesn’t seem to find itself in my bag of tricks! :) How embarassing, but thanks for correcting me guys!

    To elaborate on André’s answer, λ DNA is 48,502 bases, and at ~0.338 nm/basepair, that gives a contour length upwards of 16 μm. Any funny business that might be happening with the linking molecules (such as a slight lengthening as you increase the force on the beads) will be so negligable compared to the overall extension of the DNA molecule itself that you can safely ignore it.

    Other tricks you can do if you’re using DNA other than λ is to end-fill multiple dig and biotin labels on either end (keeping of course only one kind on either end), and my cartoon of the tether was missing the fact that the beads are coated in something like 10^5 streptavidin and anti-dig particles respectively, and so you can make a few tethers to increase the forces at either end.

    With only one tether at either end you often see the tethers break at about 30 pN of force, but with multiple tethers I’ve gotten up to about 80 pN or so before the bonds break!
  5. JK    4087 days ago    #
    Do you ever find more than one DNA strand tethered between the beads?

    When you test for a connection do you apply force using the tweezers holding the pipette fixed? I’m not sure how you would apply pN forces using a pipette.
  6. PhilipJ    4087 days ago    #
    Hi JK, good question! Yes, you can often have multiple tethers, and then the force-extension relations measured end up showing higher forces required to stretch to a certain extension than expected. Since the tethers often don’t form near each other, one DNA often gets stretched tight before the other, and you often see a bond break, which results in the extension of whatever molecules are still tethered to increase for the same amount of applied force.

    As for how I apply forces, the pipette is used to stretch the DNA that’s attached between the bead on the pipette and the bead held in the optical trap. As the bead in the trap is displaced from the trap centre by the strechted DNA, the trap exerts a force in the opposite direction, and so we don’t use the pipette directly for force measurement, but rather as the movable handle to pull on things!
  7. Anders    3466 days ago    #

    Here’s a pretty good reference for biotin-streptavidin and DIG-AntiDIG labeling:
    http://dx.doi.org/10.1093/nar/gnj016


  8. nagalakshmi    3363 days ago    #

    can i get the exact protocol or reference for the above mentioned method for obtaining 48.5kbps length lambda dna tethers?


  9. PhilipJ    3363 days ago    #

    The reference is at the bottom of the post, reproduced here:

    R. M. Zimmermann & E. C. Cox, DNA stretching on functionalized gold surfaces, Nucleic Acids Research 22 492-497 (1994).


  10. tahmineh    3316 days ago    #

    I want to find a protocol for cutting DNA’s in different lengthes.
    Is there any sources possible?


  11. Paul    3270 days ago    #

    How close do you bring the beads together to create the tether?


  12. wojtek    3266 days ago    #

    where can I find the protocol for these application? (labeling lambda DNA) with bio-dUTP


  13. Wilfried    3262 days ago    #

    Thamineh: You can cut easily DNA with restriction enzymes. See www.neb.com and their tool nebcutter. wojtek:
    As explained above, you have to use an enzyme like Klenow Exo – .
    There is nothing special for this prep. as long you know a bit of biochemistry. use the standard protocol from the Compagny (like NEB) and do not forgot:
    1) to pre-heat the DNA at 65C cause the ends of Lambda are sticky at RT.
    2) to remove the excess of non-labelled nulceotides. For that you can use dedicated columns


  14. Kurt    3242 days ago    #

    Where can I buy dig-dUTP? I have been calling around and cannot find where to get it.


  15. PhilipJ    3242 days ago    #

    Roche.


  16. KK    3239 days ago    #

    Hi Philip. How can you get a λ-DNA with 12 bases overhanging at both 5’-ends, as like your second figure?


  17. PhilipJ    3239 days ago    #

    KK – did you read the text immediately above it? :)


  18. tahmineh    3233 days ago    #

    Thank you so much Wilfried, This was realy helpful as we have not any access to a biochemist for a little help.


  19. M. Weiner    3230 days ago    #

    the picture is of phage T4, phage lambda doesn’t look like this.


  20. J.F.    2699 days ago    #

    Can I buy the biotinylated and digoxegin-labeled lambda DNA somewhere?


  21. PhilipJ    2699 days ago    #

    J.F. — not to my knowledge, and if you could, I fear it would be much more expensive than making it yourself.


  22. I.Noto    2668 days ago    #

    Will it be possible to label this lambda DNA with cholesterol? Any protocol available??


  23. Caroline    472 days ago    #

    Quite detailed information!Thanks for sharing!


  24. Rose    394 days ago    #

    Hi

    I would like to know why we are going for dUTPs instead of dTTPs? Is there something to do with the Klenow Exo? I mean like it wont incorporate dTTPs or something like that?


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