Calculating the size of E. coli chromosome?

[sheeshamagurlie]

The question sounds easy but I'm pretty confused! We conducted an experiment where we used restriction endonuclease digestion of DNA. the results after electrophoresis show the plasmid DNA in discrete bands but the chromosomal DNA is a smeared profile. The question is to design a variation of the experiment that will enable you to calculate the size of the E.coli chromosome given that you were only given two enzymes that is a 4 base and 6 base cutter.

how i hate molecular biology!

[sorin]

Hi,
i don't think that you can calculate the size of e.colis genome by cutting the chromosome with 2 enzymes. 4 base cutters cut every 256 bp on average, 6 base cutters every 4096. Given a size of the chromosome of almost 5 million base bairs you would definetly end up with too many fragments, overlapping each other so that you cannot just count them... . Usually chromosomes or very large DNA molecules are separated by pulse-field gel electrophoresis, however the e. coli chromosome is circular so you need to linearize it first...

[sheeshamagurlie]

Hi Sorin, thanks for the answer, but just wondering if pulse- field electrophoresis the only way to separate larger bands of DNA or could conventional agarose gel electrophoresis be used by using a base cutter that has a higher base count (like 4^9)?

Also, with the use of pulse- field electrophoresis, what type of base cutters are used? Can you still use the 4 base or six base cutters?

[JackBean]

the number of nucleotides the RE recognizes tells you only the AVERAGE length of the fragments, but in reality they can cut like every 20 bp or not for a million of bases... So, as sorin said, you will get plenty of fragments and you would need to distinguish them.

One idea came to my mind. If you used one enzyme for very short time, just to linearize the vector, maybe you could read the size of it. However, with such a long piece of DNA, it may not make any difference between linear and circular DNA's mobility.

[canalon]

Hi Sorin, thanks for the answer, but just wondering if pulse- field electrophoresis the only way to separate larger bands of DNA or could conventional agarose gel electrophoresis be used by using a base cutter that has a higher base count (like 4^9)?

Also, with the use of pulse- field electrophoresis, what type of base cutters are used? Can you still use the 4 base or six base cutters?

If you cut too many times, what you get is a smear because you get lots of short bands covering most o the possible sizes: no discrimination between bands. That is why PFGE is usually done with rarish cutters (not too rare, you want multiple bands, not too frequent because see above). However maybe a 6 base cutter might be okay as I have seen PFGE done with XbaI (planning to do that soon actually) and if you look on REbase you can see that the actual cutting frequency can be massively different from the expected one (see the XbaI page for example, that will tell you why XbaI on E. coli is quite good.
Another (very complicated solution) would be to do a 2 steps separation: separate large bands and purify them, then a second digestion with your mor frequent cutter. But the first step at least would require PFGE to get good separation.