Answer to the Friday Whatsit for November 5, 2010
Congratulations to Chris, April, and Linda who all guessed correctly that this is a DNA sequencing gel. It’s supposed to have 4 lanes, one for each of the four DNA nucleotides: guanine, adenine, thymine and cytosine (GATC) but the G lane spilled over a little bit to the left. The wells on these things are tiny, its hard not to spill over. Of course, spill overs and smudges make the lanes harder to read.
This isn’t a gene sequence from a particular person, this was a research sample (from an animal) that I ran a long time ago in a laboratory far, far away. These days most people don’t make sequencing gels like this, they take their samples to a facility that is dedicated to running samples through a sequencer machine. These machines are faster and more accurate than gels, making them more cost-effective in the long run.
The Sanger Method
Whether someone is running their DNA through a machine or on a gel, the Sanger Method is the method of choice for tagging the DNA and ultimately reading out its sequence. The ability to sequence DNA was such an important step in the advancement of Biology that the inventor of this method, Dr. Frederick Sanger, shared the Nobel Prize in Chemistry in 1980. He also received an unshared Nobel in Chemistry in 1958 for his work on the structure of insulin and other proteins.
The University of Michigan DNA Sequencing Core has a nice description of the Sanger method on their website. Unlike this post, they have helpful drawings. They also have a lot of scientific jargon, so take your pick.
If we were going to sequence a gene or other chunk of DNA, here is what we would do:
1) Isolate the DNA we wish to use. DNA can be isolated from any bit of living thing. Cheek cells gently scraped off with a toothpick are popular in undergraduate science labs. Isolating DNA is surprisingly easy, it can even be done at home with common household materials like dish soap and rubbing alcohol. The Naked Scientists show how to do this with a kiwi fruit.* Although what you would do with the kiwi DNA once you isolated it and got bored with playing with its gooey, strandy goodness I don’t know, the rest of the sequencing process can’t be done without specialized equipment and reagents.
3) Divide the DNA into four tubes. Into each tube, add all the things you will need to copy the DNA. This recipe will include primers (short bits of DNA from the beginning and end of the gene part you want to sequence) enzymes and free nucleotides. Free nucleotides are the building blocks of DNA, the G’s, T’s, A’s and C’s that have not been put together yet. The brilliant part (the part that netted Dr. Sanger his second Nobel) is this: to each of the four tubes, we will also add a little bit of a special, modified form of one of the nucleotides. Say this is a modified guanine (G). This modified G will do two things: allow us to see it with a special kind of light and stop the copy of the DNA from growing any longer.
4) After letting the reactions run for a while, you will have tubes full of all sizes of DNA strands. In our tube containing the modified G, we will have lots of strands, each ending at a random G in the sequence. So if our sequence was AATGCTTAGAC, in our tube with modified G‘s, we would have some strands of AATG and some of AATGCTTAG (we also have to add regular, non-modified Gs, otherwise the modified G would always stop the copying at the first G). In a tube with special A’s we have A, AA, and AATGCTTA, and so on.
5) Pour the sequencing gel and add the products of the four tubes to the top of the gel. We use a spacer to create lanes for the products to run in. Keep track of which lane contains which special nucleotide mix (in our case, the lanes were loaded G-A-T-C). The gel contains long chains of molecules called polymers. These polymers create a mesh-like substance. When we apply a current to the gel, with a positive charge at the bottom and a negative charge at the top, the DNA (which has a net negative charge) is attracted to the positive charge and moves through the mesh to the bottom of the gel. Longer strands of DNA will move more slowly through the mesh, staying close to the top, and smaller strands will move more quickly, traveling all the way to the bottom. In this way we will separate the strands by size.
6) When the gel is run, go into a dark room and expose it to a piece of film. The modified nucleotides will glow, leaving a mark on the film from where ever they are in the gel.
If we were very, very good and stayed on top of things, this whole process would have taken us about a week, depending on how much amplification we wanted in step 2 and how long we exposed the film in step 6.
7) Read out the sequence. If you go back to the picture of the gel, you will see that each lane of the gel contains a series of bands, and these bands are slightly staggered compared to the bands in the neighboring lanes. We know that the band in each lane was made by a modified nucleotide of a certain letter, so each time there is a band in a lane, that represents a known letter in the sequence (first lane G, second lane A, etc.). We also know that the longer the strand was before it ended in a modified nucleotide, the higher in the gel it will be. So if we start at the top of the gel with the highest band we can see, that is the LAST letter in our sequence. The highest band I can see in this part of the gel is a G, followed by a slightly lower band in lane A, then T, then C, then possibly two bands in G (so G or possibly GG), then back to T, then C, then GAT(?)GGAC. Reversed, this would give us CAGGT(?)AGCTGG(?)CTAG.
As you can see, reading the gel is not easy. There is uncertainly as to which letter is where because the solutions from the wells mixed together, the bands in each lane are curved and smudged, and there are imperfections in the picture that make it hard to be certain that this is the right sequence. We would have to run the experiment on the same sequence several times, and hopefully different bits of each gel would be good, so we could eventually put together a reliable sequence for part of our gene. Since most genes are longer than the number of nucleotides you can run on a sequencing gel, we would have to sequence different, overlapping parts of the gene to get an idea of the sequence of the whole thing. Sequencers help with the process because they can read out a sequence faster and with more accuracy than a person can. But even then there is uncertainty and the reactions are run several times. And in the end, it is always helpful if someone else could come along and do the same thing with the same gene, so we could get independent confirmation that our sequence was good.
Sequencing on a Grand Scale
So now maybe you have a better appreciation for the awesomeness that is the Human Genome Project. It took two independent teams 13 years (1990-2003) to sequence with 99% accuracy the 3 BILLION base pairs that make up the human genome. Now they are in the process of giving it all away, making their discovery available to everyone so that others can figure out what all that code means.
It’s a big cool world out there, but even in the gloppy, stringy goo that is the DNA in all of our cells there is mystery beyond imagination.
*Yes, the Naked Scientists are from Europe. This is why they say “washing up liquid” instead of “dish soap”. They also use metric measurements and the Celsius temperature scale. If you are not in a part of the world where these measurements are standard, consider the conversions part of your science lesson. Scientists use metric measurements too.
#Dr. Kary Mullis and Dr. Michael Smith shared this prize. Dr. Mullins is, quite famously, a surfer as well as a scientist. According to urban legend, this familiarity with the sea is what lead him to the key element of PCR. The Taq polymerase used to elongate the DNA strands is a reproduction of the DNA replication machinery of a bacterium (Thermus aquaticus) that lives at deep-sea thermal vents. Its ability to replicate DNA at temperatures high enough to melt DNA into separate strands is what allows PCR to occur in a tube in the lab, without the natural machinery that untwists DNA like we have for DNA replication in our cells.
This could be a record for number of Nobels mentioned in a single Tiny Science post. After all, the model of the DNA double helix did itself garner Dr. Francis Crick, Dr. James Watson and Dr. Maurice Wilkins the Nobel in Physiology/Medicine in 1962.