This week marks the 18th anniversary of my first attempt to sequence DNA. It did not occur to me then that my destiny would be to interpret DNA, not do the actual data acquisition.
My sophomore year in the Land of Blue Hens had been very good, and I had started working with a professor there on some undergraduate research. During the year I learned how to miniprep DNA, run restriction digests and then photograph them on a transilluminator.
I also had engaged in a grand literature search to identify all of the known DNA sequences for our organism, the unicellular alga Chlamydomonas reinhardtii. Back in those days one could not rely on Genbank for completeness -- despite there being about 10 or so C.reinhardtii sequences published, only 2 (the two subunits of RuBisCo, a key photosynthetic enzyme) were deposited. So I would borrow another professor's computer in her office (in those days, computers that moved were never sighted on campus, though luggables such as the original Compaq existed). This required a bit of coordination, as her office was not spacious enough to easily seat two & so I needed to get her to unlock the office to let me in but when she didn't need it. Out of frustration with this arrangement was born an invention: I threw together a clone of the key functionality of the entry software: you could key in a sequence once & then switch to verification mode and key it in again, with the computer complaining audibly if there was a mismatch. At the time, it never occurred to me that this was a major milestone in my career.
A second application soon followed -- I had heard that Chlamydomonas had strongly biased codon usage (it also was very G+C rich), and built my own graphical codon bias indicator. I was a bit disappointed to learn that this was well trod ground in the literature; somewhere I had gotten the delusion that the whole idea was novel.
This was all preparation for the summer. I had been accepted into the Science & Engineering Scholars program, which would allow me to spend most of the summer at school on a small stipend, in dormitory space filled with other S&E scholars as well as those in a parallel program in the humanities. I would learn to sequence DNA!
I would not be the only one learning. My adviser was a biochemist attempting to refit as a molecular geneticist, and he would be learning alongside me. But we would not be alone. The professor whose computer I borrowed was experienced in the art, and we would be borrowing her equipment & lab space. We also had a baguette of a professor (French, crusty on the outside, soft on the inside) who had written papers in the field and had also been entrusted by nearby DuPont to vet their automated DNA sequencer (it was pronounced a failure).
On Monday the 12th I showed up eager for action. My advisor laid out the gameplan: we would run the reactions today, and run the sequencing gels on Tuesday. He neatly laid everything out and then got the piece de resistance out of the freezer -- the big blue egg of 35S-labeled ATP. Within the egg's confines was the actual vial holding the radioactive compound, and the egg was nestled in it's own ice bucket. He walked me through the reactions, we ran them, and then I focused on cleaning up, scanning the bench for any loose materials.
That evening, I had one late-night activity planned. At the very end of the day I waited outside the local victualer, and when my watch marked midnight strode in & marked my newfound legal ability to order from the entire menu. Then it was back to my dorm: tomorrow was another workday.
I arrived the next day again eager for action. My advisor asked me if I had cleaned up diligently, and I nodded an affirmative. He then gently led me to an ice bucket and lifted the lid -- there, floating serenely in the melt water was the bright blue egg of 35S-ATP. I then got a good lesson in checking for radioactive contamination, but there was none. I felt guilty for wasting the 35S-ATP -- our lab ran on a shoestring, but was determined to forge ahead.
The big duty that day was to pour and run the sequencing gel. Yes, in those days there was no capillary sequencing, but rather thin slab gels. Running length meant then, as it still does now, resolution -- so we would use the 1 meter long plates.
Now a key consideration of those plates is that they need to be scrupulously clean. Any speck of dirt or fingerprint would lead to a bubble in the poured gel, destroying at least one lane and probably distorting many of the rest. Using a simple detergent, I was to clean the plates & get them ready for pouring. With help from the experienced professor we would pour the gel & then run our samples. With luck, by end-of-day we could dry down the gel and put it on film for exposure overnight.
The instructions seemed straightforward, but it soon became clear that I had been left alone with a bit of a logistical challenge. I had a standard deep lab sink in which to wash those vitreous monsters. I decided that I could wipe them down with detergent on the lab bench and then balance them over the sink for the rinse cycle.
What ensued was straight from a Road Runner cartoon, with myself as Wile E. Coyote. In mid-rinse the balance was tipped, and the far end of the plate dropped into the sink. Upon hitting bottom, the glass shattered. This altered the balance again so that the near end tipped down, delivering the freshly fractured edges out of the sink and into one of my fingers.
After the initial shock wore off, I realized that I had been seriously gashed but it was no emergency. I had a bit of first aid training, and so I compressed the wound until the bleeding slowed and then hastily scribbled a note saying I would be going to the infirmary. I then walked the mile or so to the south end of campus & had the wound attended to. Luckily, it did not need stitches (I have a potent needle phobia) but rather just an adhesive closure.
It hadn't occurred to me in the heat of the moment what a scene I had left behind. The female professor came back to check on me and apparently had quite a start: undergraduate gone, shattered glass in sink, bloodstained paper towels in the trash & a note with blood drops on it mentioning a trip to the infirmary.
The rest of that summer would not be eventful. We never did succeed in getting our plasmid to work, though I did sequence the control stretch of M13 repeatedly.
My lab career at Delaware would include another trip to the infirmary (needle stick; luckily before I injected the mouse) and another loud accident (from a swinging bucket centrifuge; carefully, but incorrectly, balanced). My undergraduate advisor would ultimately suggest that my graduate work might better focus on the computational interest I had demonstrated, not the lab manipulations I struggled with. Many years later a different group would sequence our gene, acetolactate synthase. Now, just about every gene of Chlamydomonas can now be found in the online databases, as a 3rd release of the draft genome is available; I would have never guessed at the time this would be true so soon in the future.
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