Science in the City

Of all the days I’ve spent these past few weeks in the lab, today was perhaps the most intense.  There were several factors contributing to the stress level.  First is that I was again undertaking procedures I hadn’t done before as I moved through the various protocols.  Secondly, time is getting short, and there’s no room for error if I want to get my results before returning to NYC.  The materials are super-expensive (1 ml of material I used today costs $800) and as ever, the tasks take a LOT of focus to accomplish.

After getting multiple clean copies of a selected bit of DNA made yesterday, the last step is to prepare the gene for sequencing, which will ultimately tell us the combination and order of bases (those As, Ts, Cs and Gs from your HS bio class) in each species’ version of the gene.  In the chemical processing to get to this point, however, there accumulate around one’s target gene a lot of extra stuff the machine that reads the bases doesn’t like, such as salt, bits of DNA, unused enzymes, etc.  Today I had to get rid of these, and then use flouro-labeled bases to dice up my DNA into pieces the machine can read.  You can actually see the difference in the images above of the pre- and post-cleaning DNA.  The gel on the left is streaky with diffuse, multiple bands, indicating objects of many sizes in the mix.  After, on the right, one can see much less streaking, and nice tight bands of uniformly sized material.

To get to the final image, the plate on the right, I had several more steps to go.  First, I made a dilution of each individual tube, two times, based on my prior day’s result to get the same amount of DNA in each.  I was leveling the playing field.  Next, I added to each sample a chemical that would mark and cut the DNA into lengths with a tag on the end showing what the last base was (**I’ll get back to this bit for the technically inclined.)  Then I had to create a foam, put it into a special tray with little funnels in it, and then run my DNA through it to get all the ‘junk’ out.  Simple as it sounds, it took me from 9:45 in the morning to 6 pm in the evening without pauses.  The work takes incredible focus, since often one is moving 1 critical microliter of sample from one tube to another.  That’s .000001 liters.  A mere capful of soda would contain a couple thousand of them!  So it’s easy to make a mistake, especially when you’re doing each step 100 times over.  My hope is that the sheer number of tries and redundancies in my sample set mean I get a complete result, in spite of myself!  At the end of the day, an index was created (keep track of what sample ended up where was in itself a Rubik’s cube-like task) before it went to the sequencing lab.  I am told that we are first in the queue for Monday, and that we should, with a little luck, have something to celebrate by lunch time!  For all but those interested in the principle that operates the sequencing reaction, see you again, online!

**Extra credit for the technically curious among you:

The reaction that marks and cuts the DNA is interesting.  The target DNA, called a template, is mixed copying enzymes and  the raw materials of DNA, including the individual bases.  Sprinkled among the normal bases in the mix are occasional versions that are special for two reasons.  First, they have a ‘marker’ attached, which can be read by a sequencing machine.  They’re like the others, but they’re special – sort of like the powerball in the lottery, they count for a bit more.  Secondly, besides from being marked and machine readable, they also can arrest the enzymes’ copying of a particular segment of DNA.  So – mixing a few of these into the normal DNA copying process yields lots of little pieces of DNA that got copied complete to a certain point before the labeled base was inserted where a normal base would go.  This event halts the copying of that segment of DNA, leaving a clue we can use to piece the puzzle together.  Putting thousands of such copies arrested at different points through the sequencer, we can tell two things.  One is how long the piece is, and second is what ’special’ base was the last one used which froze it.  With many such bits of info, we can puzzle out the actual combination.  

For example, if we had five fragments with known lengths and end bases:

1. 1 base long, ended in ‘A’

2. 2 bases long, ended in ‘A’

3. 3 bases long, ended in ‘C’

4. 4 bases long, ended in ‘T’

5. 5 bases long, ended in ‘G’

Then we could infer the gene base order is ‘AACTG’….just like an acrostic poem, you read it from top down to get the hidden meaning!  That (in simplified principle) is how the sequencer works.