One of the analyses I proposed in the DNA-uptake grant proposal is designed to find out whether uptake signal sequences are unusually flexible or bent, by testing whether presence of a USS helps short DNA sequences bend around into circles whose ends can be joined by DNA ligase.
Because competition for grants is very tight this time around, we want to be prepared to submit even better proposals in September if the ones we submitted two months ago aren't successful. This means we want to have lots more preliminary data showing that the experiments we propose will actually work. So one of the post-docs has been working to get preliminary results for this circularization experiment.
The DNA fragments should be about 200bp long; shorter ones usually can't circularize at all, and longer ones have enough flexibility that the ends readily bump into each other. Even for fragments in the right size range, exact length is critical because of the 'polarity' of the ends of the double-stranded DNA. Each 'end' is really the ends of two base-paired strands, one ending in a 3'-OH and the other in a 5'-P. When the ends do bump into each other, ligase can only join them if a 3'-OH is aligned with a 5'-P (the bond must be a 3'-5 connection, not a 3'-3' or 5'-5'). Because the two strands of the DNA wind around each other every 10.4 base pairs, the length of the DNA fragments must be approximately a multiple of this length so that the ends will meet in the right alignment. The strategy is to use PCR to synthesize fragments of the right length, and she designed two sets of primers, giving fragments of 208 and 260bp (my notes say 260 but I think I have it wrong as this seems too long, unless it's the positive control).
The ends created by the PCR process are not easy to ligate, because they have inconvenient incompatible tails, so her primers include sites for digestion by restriction enzymes. Cutting both ends of the PCR product with the same restriction enzyme will generate compatible 'sticky ends' that can base pair with each other. The base pairing will hold together any ends that do bump into each other in the right orientation until ligase can seal them together permanently.
Sounds good so far. But there's one more factor. The circularization reactions must be done at low DNA concentration to decrease the frequency of ends of different molecules bumping into each other. This intermolecular reaction has 'bimolecular' kinetics, meaning that its rate depends on the DNA concentration. In contrast, circularization has 'unimolecular' kinetics, and its rate is independent of the presence of other molecules and thus of DNA concentration. Doing the reaction at low concentration is easy (need less DNA), but detecting the results of the ligation is hard, because small amounts of DNA (linear or circular) are difficult to see in the gels used to separate the different conformations that result from the ligation.
The solution is to label the DNA fragments with 32-P, making even very small quantities easy to detect. The post-doc followed a published method for labeling DNA for these experiments, which should put a 32-P at each 3' end of each fragment. The fragments are first cut with the restriction enzyme, purified using little spin-columns, and then incubated first with a phosphatase, to remove the non-radioactive P from each 3' end, and then with the 32-P nucleotide and a kinase, to put the hot phosphate on. Then the fragments are purified again.
Initially I was concerned by the low recovery; only about 10% of the input DNA was recovered after all the intervening reactions and clean-up steps. After a bit more thought I became more concerned by the labeling reactions, mostly because I've always found phosphatases to be nasty treacherous enzymes that don't know when to stop. If the phosphatase removes more than the single terminal phosphate, the fragment will not be circularizable even if the kinase then does its job correctly. Furthermore, any fragment that the kinase misses will also not be circularizable, even if the phosphatase has behaved itself. Even if one end is processed correctly, any problem with the other end will still prevent circularization. In principle these problems can be controlled for, but any experiment-to-experiment variation will invalidate the conclusions we're hoping to achieve.
Luckily, once I started worrying about these issues I realized that we can eliminate both the recovery problem and the phosphatase/kinase problems by labeling the DNA internally rather than at its ends. So the new plan is to add a labeling step after the PCR reaction. This will be essentially one extra PCR cycle, this time with one radioactive precursor nucleotide added to the mix. The resulting DNA fragments will then only need to be digested with the restriction enzyme and cleaned up once.
When the post-doc gets back from her visit home, we'll still need to solve the problem of why the gels run so oddly, but at least we'll have enough labeled DNA to do lots of tests. The gel problem may be related to the high concentration of ligase needed in these reactions. The standard ligase stock is purchased in 50% glycerol at the low concentrations needed for cloning reactions, and the circularization reactions use so much ligase that they are about 25% glycerol. I'm hoping we'll be able to buy a high-concentration ligase stock, rather than having to make our own ligase....
Real tough guys - lichens
3 hours ago in The Phytophactor