One of the first molecular biology techniques I learned as an undergraduate was restriction enzyme mapping. It's simple and beautiful; at the end you have neat bands of orange glowing in the darkroom.
Molecular biology involves a lot of incubations, giving one time to read, think or work on other projects. An easy way to pass some time was to pull out the New England Biolabs catalog and browse. NEB sells a lot of reagents, but their selection of restriction enzymes has always been a key point. In addition to the enzymes themselves, there were the restriction maps of common vectors in the back.
Restriction enzymes are simply amazing, nature's gift to molecular biology. Each enzyme recognizes a short DNA sequence with incredible specificity, cleaving only on or near the appropriate sequence. All sorts of interesting variations on the theme exist. Some are blocked by methylation of nucleotides in their recognition site, others require methylation. Some cleave in a region of precise length but undefined sequence between their recognition site; some cleave a select distance away, and a few clip out an island of DNA centered on their recognition site. The taxonomy of these enzymes simply grows & grows as new variants are identified.
During my graduate years I didn't work with restriction enzymes, other than one concept that never got beyond the idea stage. At Millennium it was totally outside my scope.
But now, in the synthetic biology world, I get to play again. I'm again browsing through the lists of enzymes, though now I do so with REBASE. How many other databases are labors of love by a Nobel laureate? As an undergraduate some of those outside and island cutters seemed to be oddities; now they are opportunities.
In particular, the Type IIS restriction enzymes, those which cut adjacent to their asymmetric sites, have really moved into their own due to their utility in manipulating DNA. By ligating a IIS site to unknown sequence, one can clip out a short tag easily sequenced, such as in SAGE. In synthetic biology, designing IIS sites into a sequence can be used to generate a huge variety of sticky ends, yet also leave no 'scar' in the final sequence.
Of course, one can never be satisfied. Enzymes with very rarely occurring sites are useful for a lot of genomics research, but very few restriction enzymes with long (and therefore rare) recognition sites have been found. There are only limited numbers of methylation-dependent enzymes, or IIS enzymes. Not only do enzymes vary in their recognition sequence, but even enzymes with the same recognition sequence can cleave at different positions (using different enzymatic mechanisms), which can be useful -- but for many sites only one cleavage pattern is available.
Ah, no matter how impressive the toy chest, we still have a wish list!
2 comments:
I know they are useful tools and all, and nobody questions, for example, why the Swiss Army needs a spiffy knife. But I've seen very little speculation (apart from my own, ages ago) on the biological function of restriction enzymes.
What do you think they are for (apart from being for molecular biologists)?
I'm no expert, but the two general classes of evolutionary explanation are
1) Defend yourself against foreign genomes (e.g. viruses).
2) Restriction/methylation systems as selfish genetic systems which once acquired, can't be evicted.
The wide variety of different RE architectures hints that there must be some interesting selection for them -- they keep being invented. Also, there are various papers on converting restriction enzymes to nicking enzymes or vice versa.
Here is one review, with a strongly protein structural angle.
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