Tag Archives: science

How to Achieve Your Dreams

sky-clouds-above-clouds-wallpaperThe following is a response to a question on Quora. I didn’t quite read the question correctly, but I like what I came up with as an answer. I have no definite expertise on any of this, but I have learned a few things that may be useful for somebody.


dmaicIf you’re serious about accomplishing your dreams, there are some tools that have been developed in the business world that can help. I’ve been involved with some Six Sigma projects, and I like the model of DMAIC for the most part. That is Define, Measure, Analyze, Improve, and Control. These sound boring, but they’re really not. Let’s go through them.

Define
The first step of accomplishing your dreams is to have realistic dreams. This isn’t giving up, this is building up. If you want to fly, great. I’m not going to say you can’t fly. But what exactly do you mean by flying? Is flying an airplane going to work for you? No? How about skydiving, that’s kind of like flying, or hang gliding? Do you actually want to be like a bird, or do you want to be superman?

Already, while we started off with something that seems unattainable, we now have several options that are much more attainable. One of the things that keeps you back in this step is fear. Your dream of flight is nice, but what if you don’t like it? What if it’s dangerous? What if it seems less beautiful? These what-ifs can hold you back, but they shouldn’t be ignored either. Make a list of these fears along with any other risks, because its entirely possible that you actually don’t want to accomplish this particular dream. Next to these risks, you want to list the benefits of accomplishing your dream. What will you gain from it. Why do you want to accomplish it? It could be that while your dream involves flight, what you really want is a feeling of freedom, and there might be better ways of doing this. If you think of some, great, make another list of risks and benefits for this new dream. Does it look better than what you had before? You keep at this until you have something that matches your desires and yet still fits with what can occur in reality.

If, at this point, you still don’t see how you can accomplish your dream, then look for ways to get closer to accomplishing it. If you want to be Superman, maybe look into jet packs, physical fitness, rescue work, or space travel. Depending on what it is about Superman that you most want to be, learning about these subjects might not get you there, but it will get you closer. The idea here is to bring your dream closer to reality by bringing reality closer to your dream.

Measure
weight-scale-400x400-300x300How close are you to your dream today? The easiest example of this is weight loss. If you want to lose 30 pounds before next Thanksgiving, you need to monitor how you’re doing to know if the diet you are on is working or not. Even if it’s something you dread, you have to get on the scale and check, otherwise there is no point in dieting. You can get more sophisticated in this step for more benefit. You can make a graph of your progress for example and a list of all the foods you ate each day and any exercise you did. This way if there is a large dip or a peak, you can see what might have caused it. Weight loss is nicely quantitative, so measuring is easy to do.

The picture to the left links to a blog about not letting scales control your life. This is a common sentiment, and I can understand where it comes from. I think it’s a completely wrong way of thinking about things, but I understand it. If you aren’t having too much success accomplishing your dreams it can be disheartening to measure how far you’ve fallen. This is because humans aren’t robots. If you aren’t doing well in your progress, you need to either move on to the next steps (analyze and improve) or reconsider the previous step. Is loosing weight really what’s important to you? Could it be that you’re really worried about your fitness? Weight may not be the best way to measure that. If you stress eat, you might measure progress by recording times of stress and how you coped. Or maybe whatever is causing you stress is the problem. Putting serious effort into fixing that might be the best bet. You are never going to be able to levitate yourself. That’s depressing. But being able to do a pull up is close. Even Superman started with tall buildings. But whatever you’re goal you HAVE to measure your progress toward it somehow if you’re serious about accomplishing it.

For your more qualitative goals, like flying or perhaps owning your own business, you might need to make a journal dedicated to the goal. You can probably come up with many little goals and you can note your successes and your set backs in a journal as you experience them. The point is to have a record of what works and what doesn’t so you have some guidance in the next step.

Analyze and Improve
If your diet isn’t working out, change it. If you feel like you’re not getting anywhere, move. You do NOT want to be consistent if you are unhappy. A foolish consistency, as Emerson said, is the hobgoblin of little minds. Although you want to make changes that are likely to work and that aren’t too dangerous, remember it’s okay to make mistakes. You are, in fact, required to make mistakes in order to know what does not work for you. There are different kinds of writers, different kinds of digestive systems, you may, in fact, be from the planet Krypton. You can’t just follow someone else’s plan.

It is very important, however, to continue to measure your progress. You might think it’s a good idea to eat nothing but soy products in your diet, but you might find out that you actually gain weight (because, surprise, soy can be fattening!) Give it some time so you can be sure of how things changed, but if things are going worse, change your system back to how it was before if possible, or make another adjustment if its not.

houseflyI’m going to digress here for a moment to talk about house flies. If you watch a fly fly, you might notice how randomly it moves. It buzzes around your sandwich quite a bit yes, but then it takes a trip to the window and the to the lamp shade and back to your sandwich again. The actual motivations and causes for the complicated behavior of a fly is complicated, but one possible way of explaining it is as a modified random walk. A random (or drunkard’s) walk is one in which a moving object moves in a random direction for a random amount of time. The fly does just about the same thing, except it has memory, sight and smell, signals that make it prefer certain directions over others. As long as the good signals are getting stronger, the fly will keep going in the same direction, pretty much, but if there’s a bad signal that’s getting stronger or the good signals are getting weaker, it sort of tumbles in the air and flies in a new, but still mostly random direction. This is rather inefficient, but it works. If you are near a sandwich, and you get further away from it, the signal goes down. You then go in a different direction and maybe this is also leading away from the sandwich so you change directions again, and now you are going toward the sandwich again so you keep going. I should say that the fly is a bit (a LOT) more sophisticated than I’ve described here, and, while a fly does act this way somewhat, this behavior is more like how bacteria move (Howard C. Berg has written a lot of interesting work in “random walks”). The point is, that it doesn’t matter so much what you adjust, or in what way you adjust it; what does matter is how often you adjust it and how closely you monitor your situation.

If things are progressing well, don’t mess with them! But if things aren’t going anywhere, some kind of change is in order. If things are getting worse, than a change is not only a good idea, but an urgent one. Don’t let fear keep you from a better life. You might be in a situation where you don’t have a lot of options, but even if you only have two paths you can go on as Led Zeppelin says, “in the long run, there’s still time to change the road you’re on.”

Control
ControlThis step has two phases. While you’re still attaining your dream and making adjustments, you will probably find it helpful to establish some rules and guidelines. For example, jumping off a building is not an acceptable means of learning to fly. In weight loss, you might find that day to day, your weight varies by about a pound or so there’s no reason to stress about an increase unless its more than that (stress->despair->ice cream so limiting stress is also important). You may also have some go-to adjustments for when things go wrong, such as doing more exercise or taking a quiet moment to watch the birds when you’re feeling down. These guidelines that you develop on your way to your goal are the first phase of control.

The second phase of control occurs after you’ve attained your dream. So you’re successful. Now what? Well, the answer to that question is usually that you want to make sure you stay successful. A lot of the guidelines you came up with in the first phase will work in this second phase, but there may be a few things you need to do differently. If you get a job as a pilot, for example, you need to review all the safety procedures even though day-to-day you may not need to know them. You also need to keep an eye out for new technologies and if necessary train yourself on them so you don’t become obsolete. Once you lose the weight, you have to stay vigilant to keep it off, and you may have to employ different strategies as you age or go through other life changes.

As you may have noticed, all these “steps” overlap, and turn back on themselves like eddies in a river. You could in fact just as easily start with Control, and then notice somethings out of whack and move to Analyze and Improve and then Measure, and then find out what exactly the problem was at the end of the whole process (Define). That’s more or less what happens when police make an arrest. Perhaps it would be better to call these phases or even aspects of goal setting, but I think if you’re looking for a way to start accomplishing your dream, defining your dream is a good place to start. If you’ve got your dream well-defined, measuring your progress is the next thing to try. Then adjusting things as needed, and finally controlling them once you have things pretty well established. They build on each other nicely that way, and besides, that’s how the business world groups them.


Note: This post used pictures from the following websites. Please visit them and consider purchasing any products they’re selling.

http://www.jeesukkim.net/velocity/

http://www.sixsigmadaily.com/what-is-dmaic/

http://blogcritics.org/how-heavy-does-your-scale-weigh-on-you/

http://www.qpm.ca/Pests/House-Fly-How-to-Kill-Exterminate-Get-Rid-Eliminate-Pest-Control.html

http://www.mwultd.co.uk/services/part-exchange/control/

Author Attribution

In a scientific paper, the area under the title where the authors are listed is more important than you might think. Obviously, each person listed as an author has (probably) contributed to the paper in some way. There are also often asterisks and other symbols leading to footnotes at the bottom of the first page that tell you which university each author is from. That much you could probably figure out on your own. But there’s a whole snarl of politics involved in how the authors arrange their names. The first name on the paper is usually the person who did the most work on it. This person is typically a graduate student or a post doc (short for post-doctoral, a post doc is someone who has gotten their PhD, but is not yet in charge of a lab).

The first author is not always the most important author in the list, however. By this I mean that if you google their name, (or use a service that lets you search through scientific journals, such as Pubmed)  you might not find much. The head honcho working on the paper is actually more likely to be the last author listed. Google the last author’s name, and you can probably find the website of the lab that worked on the paper fairly quickly.

The head of a lab is often called a PI for principal investigator. You might think of them as a boss, but it might be more accurate to think of a PI as a manager or agent who has his or her fingers in everything, trying to get it to work. A PI can have a lot of power, depending on how much money his or her lab can generate, but they rarely have the power of a CEO. Even in the case of labs that work in the private sector, CEOs don’t have the time to write scientific papers and PIs don’t have the time to take care of high level corporate decisions.

A PI has to keep track of all the scientists he or she is in charge of and find resources to ensure that the lab can continue operating.  Finding resources means writing innumerable grant proposals and promoting research by submitting posters and giving talks at conventions. PIs don’t generally get to do the actual experiments much at all, but they have to know everything that’s going on, and the work couldn’t be done without them. So to make a gardening analogy, the last name is usually the one responsible for providing the soil and seed for the paper, while the first name is the one who waters it and makes sure it’s healthy.

The other names in the author area may be of varying importance. In papers written by an extensive team of scientists there may be hundreds of names in the author area, some of which might not even have had anything to do with the work published. This is because by being part of a team the scientists agree amongst themselves to share credit for any discoveries that the team makes. Which sounds nice, but it is important not to make the mistake of thinking that everyone listed agrees 100% with what’s in the article.

You might also have people listed as authors who might not have even met the other authors. This can happen in situations where someone gathers data, but leaves the organization before anything can be done with it.

There is also sometimes space in a paper for people who have helped, but may not have done enough to warrant being a full author.  Unfortunately because of the large egos involved in science, sometimes someone who really deserves to be a full author gets shoved into this space. You might imagine a beleaguered graduate student, passionate for his subject, yet perhaps socially inept, toiling away at his experiments until he finally has enough data to reach a conclusion. He writes the paper, following all the guidelines and diligently proofreading for grammar mistakes. He gives his paper, his baby, to his PI, thinking now, finally, he will get the recognition he deserves. Only instead of being the first author, the PI puts his friend in as the first author, puts his name as the last author and gives the grad student an honorable mention. The movie Dark Matter is a chilling depiction of this scenario.

There are several organizations, including the Office of Research Integrity (ORI started in June 1992), that are in charge of confronting false author attribution as well as several other forms of misconduct. This is good, but on the downside the very fact such organizations are necessary speaks to how prevalent the problem is. A survey conducted by Eastwood, S et al (referenced here) , where postdocs and new PIs were asked to self-report their conduct showed that 41% said they would list someone as an author for being a friend or for funding them in the future. And the ORI reports that the number of reported cases of general misconduct (of which author mis-attribution is a part)  has been rising, albeit with several large peaks and valleys from 159 in 1996 to 286 in 2010.

Does this mean that all scientists are crooks? No. Does this mean science can’t be trusted? Well, to an extent, yes. But science shouldn’t be trusted. Any information a paper relates should be tested and retested independently not just because sometimes scientists misbehave, but because the information may be incomplete or inaccurate.

If popular media could understand this a little better, I think there would be a lot less confusion about the various studies that come out. Next time you see a news report that reads “Scientists say…” you should ask “Which scientists?”

Tracking Down Scientific Sources


You may have encountered this problem yourself if you’ve ever tried to track down the source of some study from a blog or a piece in a popular magazine. You go through an exhaustive google search until you finally find the article you’re looking for. You click on the link for it, anticipation mounting as you wait for the page to load up. And then, instead of seeing the full article, you see the abstract (or summary) of the article and a little box that says that you can look at the full article if you purchase it for $30 to $40. “Screw that!” you say, “I could buy ten whole magazines for that much!” And unless you have a great deal of patience, that’s where your search ends. And even with patience you might not ever find what you’re looking for without shelling out cash.

Journals are aware of this problem and are finding ways of making it easier to get access to articles. Several journals, such as PLoS One, post all of their articles free online. Others such as Nature and Science, still keep most of their content as pay only, but offer a few articles online. But here are a few tips to get past the pay wall next time you see it.

 (1) Pay the money. Just for completeness, I’m putting this here. If you think of it as a donation for science, paying the fee for the article might not be such a bad idea.

 (2) Become affiliated with a university library and use their journal access. You might be able to use your public library for this as well. Nearly all libraries have stores of journal articles in them, but public libraries tend to focus on popular and literary magazines, while university libraries are more likely to have the more obscure scientific journals. University libraries also usually have a site through which you can access almost  any scientific journal you want to find.  The “almost” is stressed though, because the way that a library gets access to a journal is by paying for it, and if a journal is small or in a strange niche, your library may not have deemed it that important.

 (3) Copy the title of the article and search for it online. This is where you start to be sneaky. Google throws a lot of links at you that you have to cull, but you can narrow your search by copying the exact title of the article and putting it between quotation marks. Often, even though a journal won’t let you see an article, the scientists who helped write it are more than happy to let you see it, and so they might post it on their website. Or it might show up on someone else’s website. Another way you can narrow the search is by using “filetype:pdf” in the search box. If you do this, google will find only pdf files for you, and most scientific articles are in .pdf form.

 (4) Search the name of the last author of the paper. It may seem strange, but the last author of a scientific article is often the most important person involved with it. If you search their name online, you can probably find the website for their lab. The website for the lab usually has a list of publications. You might get lucky and find a link for the article you’re looking for.  Even if you don’t, you might find an article that’s extremely similar to the one you’re looking for that is more available. Such an article might be even more helpful than the one you were trying to find. This can happen because often the information published in smaller articles amounts to an addendum or a further confirmation on research the lab posted earlier.

Along these lines, if the thing you’re looking for relates in any way to human health, you might go to the site PubMed, and seach for it there. There’s a small chance you might find a way to view it from doing this, but more than that, it’s much easier to find articles from the same authors or in similar subjects there. Even without you asking it to, Pubmed will list articles similar to the ones you search for, and again, they might actually give you more or better information than what you were looking for in the first place.

 (5) Look up the contact information for one of the authors and ask them for a copy of the paper. Usually the contact information is written right on the abstract for the article. At most you’ll have to do some googling to get it. Most the time, scientists will probably be happy to give you a copy through an email, though they might be curious as to what you hope to do with it. So if you’re very keen on reading an article without paying the amount you’d pay for a DVD for it, or if you found an article that you can’t even pay for, just asking one of the people who wrote it for a copy nicely cuts the Gordian knot.

Hopefully, these strategies can get you past the pay wall, and let you read the article you’re looking for.

Gels

http://bit.ly/uMnIip

You might think that gels are just things you put in your shoes or in your hair. You might just think of Jello when you hear the word. But gels can do a surprising number of things and can be used in a lot of interesting ways.

First off, what exactly is a gel? The part you know is that a gel is basically a material that’s somewhere between a liquid and a solid. It holds its shape like a solid, but jiggles and deforms as if it were a liquid. What you probably don’t know is that this strange state is caused by a battle between several different forces, some of which try to pull a gel apart, while others keep it together.

Most gels are made up of long stranded proteins or sugars that have a strong negative charge in environment of neutral pH, a situation similar to what  molecules of DNA experience. However, unlike DNA, gel molecules can cross link to one another if they are heated and then cooled. Because of these crosslinks, as much as it would normally take a lot of force to bring the negatively charged molecules together, the crosslinks hold them so they can’t disperse. It’s like a small crowd of people that decide to hold one another’s hands. If they then try to get as far apart from one another as possible, they will form a fairly stable circle. The spaces between the molecules in a gel are usually filled with water or some other liquid medium, giving them their liquid-like properties.

Because gels have these spaces, they can be used as nicely uniform filters as in polyacrylamide and agar gels used in electrophoresis. You can also make aerogels or xerogels by quickly evaporating the liquid inside a gel without allowing the gel structure to change. The resulting materials are light weight (because they are mostly air) and have incredibly large surface areas.

http://bit.ly/vyKHu3 : A picture of the structure of a dried hydrogel (or xerogel)

To illustrate this last quality, imagine a solid centimeter-wide cube. The surface area of the cube would be the area of one face of the cube times 6, or 6 square centimeters. Now imagine the cube is broken up into eight cubes each a half a centimeter wide. The surface area of this arrangement is going the six times the area of all the faces on all the cubes, or 6 times 8 times ¼ square centimeters, or 12 square centimeters. That’s twice as much surface area as before, even though the combined volume of the cubes hasn’t changed. If you imagine this happening several more times, you can begin to see what a staggering amount of surface area an aerogel can have.

Why is surface area important? If something has a large surface area, dust and other materials have more opportunities to land on it. This is why if you look at air filters they’re made up of dense meshes of stringy material. Air filters are, in some ways, a primitive form of aerogel.

Another neat thing that we can do with gels is use them as sensors. Because the space in between the molecules of gels can be filled with many different solutions, and the molecules themselves can be engineered to have different properties, gels can be made to drastically change their properties with temperature, in the presence of different chemicals, or when the pH changes. Gels that do this are sometimes called smart gels. Gels can also be made from protein fragments to provide a scaffold for specialized cells, allowing them to be used for a variety of applications in medicine.

Probably the most amazing gels however are living cells and tissue. If you think about it, almost every tissue in your body is made up of a network of molecules connected together and filled with liquid. You are essentially a conglomeration of gels.

How to Make a Mutant: Transgenesis

(this creature does not exist)

Even though at some level, we’ve been making mutants for hundreds, even thousands of years through selective breeding and agriculture, these aren’t the kind of mutants we think of. We usually expect something bizarre and alarming that happens almost immediately. Like the Incredible Hulk turning green, or turtles becoming sentient, bipedal, and proficient in martial arts. We get a little closer to that sort of thing with environment sensitive genes.

Biologists usually first learn about these genes in bacteria, with the lac-operon system. The lac-operon system is an arrangement of proteins and DNA that essentially lets bacteria adapt to their environment. Bacteria like to eat sugar, and they survive the best on glucose, the simplest of the sugars. If glucose isn’t available, they can eat other sugars, like galactose or lactose, but that requires more machinery and the bacteria don’t want to rev up those machines until they know they have to. How the lac-operon transcription system works involves the use of two proteins, one that blocks the expression of the lactose-eating gene lac-x and one that amps up the expression. When lactose is present, the blocker protein can’t work, and the amp-up protein only binds if there isn’t any glucose. So with this system, a bacterium can shift to eating different sugars like a car shift gears.  This is something of a simplification of the process, but the upshot is that while the bacterium’s DNA remains the same, it’s expression is altered significantly by the environment.

Similar situations occur in more complex animals. Researchers often make use of temperature sensitive genes in fruitflies to create flies that are genetically identical to a control group, but have a protein that works differently due to being exposed to higher temperatures while forming inside the egg. In nature, this happens with many cold blooded animals whose sex is determined by the temperature where their eggs are kept.

These environmental affects control the expression of genes, and not necessarily the genes themselves, still, this isn’t as trivial as it may seem. The well-known statistic that our genes are 96% similar to chimpanzees’ is only true when we don’t count the so-called junk DNA that doesn’t seem to directly code for anything. But while this DNA doesn’t seem to make anything, recent evidence has shown that it may have an effect on how things are made or when they’re made. In other words the obvious difference between us and other animals may have more to do with how our genes are expressed rather than what genes we have in the first place.

”]Scientists can also insert known genes into the genomes of animals to create transgenic creatures. For example the gene that produces the fluorescent protein GFP in jelly fish can be inserted into the DNA of bacteria using electroporation, a technique where a electric field is used to create pores in the cell membrane of bacteria, which allows foreign DNA to enter. This can also be done through abruptly increasing the temperature, which forces the bacteria to open pores in their membranes to adapt to the situation.

Bacteria will incorporate DNA into their genome, and as long as the new DNA doesn’t interfere with their ability to survive and reproduce, the bacteria will quickly grow in number, providing a way of naturally increasing the amount of foreign DNA available. Scientists can then use  enzymes to cut out the gene that they want from the bacteria and insert it into the genome of a more complicated organism through several methods

Lab workers can insert DNA directly into the nucleus of a stem cell, for example, or simply allow the cell to take in the DNA on its own. Alternatively, scientist might use a virus that has had all its DNA replaced with the foreign DNA so that it can be injected into the cell without direct human supervision.

Optogenetics Experiment in Mouse (Source: MIT)

One of the most dramatic examples of transgenic research involves the use of a protein called channel rhodopsin. This protein is related to the protein that detects light in the cells of our eyes. When the gene that produces the protein is introduced into the neurons of a mouse, and a light of a certain color is show on the neuron through LEDs or  fiberoptic cable implanted in the mouse’s skull, it can cause the neurons to fire, directly affecting the behavior of the mouse. Not only that, but the channel rhodopsin can be altered to respond differently to different types of light so that blue light might cause a neuron to fire, while orange light might cause it to stop firing. This system has opened up an entirely new field of biology called optogenetics and researchers are currently finding ways to use the techniques to help sufferers of all sorts of brain diseases from Parkinson’s to Alzheimer’s.

So real mutants might not fly or shoot laser beams out of their eyes, but in many ways they’re even cooler than that.

Reading your Blueprint: Genome Sequencing

Logo of the human genome project

In this post I’ll go over the third and most recent method of identifying people through DNA:  looking at their genome. A genome is the sum total of all of the genetic information of an individual organism. If using electrophoresis and PCR is DNA fingerprinting, determining someone’s Genome is writing their DNA biography. It took some ten years for the human genome project to be successful in producing the genome of a human,  a testament to just how much information the genome contains.  Nowadays, of course, scientists and technicians can determine someone’s genome in much less time (about 4 weeks with one machine in 2009), due to the incredible advances in technology we’ve enjoyed, but how did we even get started?  DNA fingerprinting is great if you already know what you’re looking for, or if you want to compare samples of something to something else, but how do you get from that to figuring out every piece of genetic information about that thing?

Well the place it starts is with DNA sequencing. DNA is made up of nucleotides that code for various proteins. About the best thing we could hope for then is to know the sequence of these nucleotides on a sample of DNA so we can know which proteins it will code for. DNA sequencing is the process of determining this sequence.

You might recall that with DNA fingerprinting, restriction enzymes cut up DNA into fragments at specific nucleotide patterns. Those fragments split and replicate again and again through PCR, then a researcher will push them through a gel by electrophoresis, which forms bands on the gel at different levels. The closer the band is to the other end of the gel, the smaller the fragments of DNA are inside that band, and you can compare different samples by seeing where the bands show up when you subject them to the same restriction enzymes.

DNA sequencing takes this a step further. The nucleotides that make up DNA can be in two forms, deoxynucletide-tri-phosphate (dNTP) which is the usual version, and DIdeoxynucleotide-tri-phosphate (ddNTP) which has an extra hydrogen on it that keeps any more peptide bonds from forming. This means that if a DNA strand is elongating, and a ddNTP attaches, instead of a dNTP, the DNA can’t elongate any more. It’s done, terminated, its story is over.

Okay, so what? How does this get us to DNA sequencing? The way we get the sequence of a piece of DNA is  by labeling the DNA fragments by attaching a fluorescent or radioactive marker to either the ddNTP, the primer (which starts the elongation of a piece of DNA) or the dNTPs that make up the rest of the DNA.  Then you can separate each sample into four separate containers, and in each container you add in a different kind of ddNTP. Remember there are four nucleotides found in DNA, Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). Each of these has a terminating, ddNTP form that we can add to a sample. So now you have four samples and each one has a different terminating nucleotide added to it. What this means is that wherever that nucleotide normally appears in the DNA sequence, the DNA well stop elongating there. So for example, lets say we have a piece of DNA with the sequence

CACGATTCGA(10 NTPs)

In the first sample we add the adenine ddNTP so in that sample we’ll have DNA fragments that look like this:

CA*(2NTPs)

CACGA* (5NTPs)

CACGATTCGA*(10NTPs)

Because the ddNTP will attach at different points as the DNA elongates during PCR and each possible fragment size will be amplified equally, all of these fragment sizes will be available in the amplified sample that is put through electrophoresis. These different fragment sizes will then form different bands based on how large they are.

If you put all the ddNTP additions together on a gel , the example above would look something like this:

|——A—–|—-T—-|—-C—-|—-G—-|

10–BAND-|—-0—-|—-0—-|—-0—-|

9—–0—-|—-0—-|—-0—-|–BAND—|

8—–0—-|—-0—-|–BAND—|—-0—-|

7—–0—-|–BAND—|—-0—-|—-0—-|

6—–0—-|–BAND—|—-0—-|—-0—-|

5—BAND—|—-0—-|—-0—-|—-0—-|

4—–0—-|—-0—-|—-0—-|–BAND—|

3—–0—-|—-0—-|–BAND—|—-0—-|

2—BAND—|—-0—-|—-0—-|—-0—-|

1—–0—-|—-0—-|–BAND—|—-0—-|

 

Looking at this result, a technician can tell the exact sequence of the DNA by simply putting the nucleotide where it’s base shows up in the sequence: 1:C,2:A,3:C… and so on.

This is pretty nifty, but this example only deals with a DNA fragment ten NTPs in length. A human genome has DNA that is billions of NTPs long. How in the world can a genome get sequenced in any reasonable amount of time?

The answer comes in three parts. The first trick is to automate the process, so that a researcher doesn’t have to guide each process along by hand. The next trick, related to the first, is to conduct sequencing experiments in parallel. In other words, you want to have several sequencing experiments going on at the same time. The reason why this second technique is related to the first is that the way to do this is to have an array of wells with samples and the required chemicals inside them, then have a machine which deposits a controlled amount of required enzymes or other materials to each sample at the same time. The machine can then heat each well and allow it to cool as needed for PCR.  The final trick is to break apart a long strand of DNA into much smaller fragments and then sequence those fragments randomly, rather than try to do fragments in the order they appear naturally this technique is called shotgun sequencing.

You might wonder how, after all these random fragments are sequenced, can researcher’s put them back together in the proper order. The way this works is similar to a jigsaw puzzle. In a puzzle you may have several pieces that are obviously part of the sky, say and other pieces that are part of other separate areas. If you have several sequences, some of which overlap each other, you can put them together by recognizing that some of them are part of a recognizable pattern. You can put the sequences from fragments with the same pattern together and proceed through the whole genome that way. Obviously, if you were to try to do this by eye, it might be very time consuming, but with the help of computers the situation becomes manageable. So much so that the newest forms of genome sequencing use much smaller fragments that are only few base pairs long, allowing them to sequence all of them in parallel very quickly. A computer can then use statistics to predict where each fragment would show up. This method produces more errors, but makes up for it by the amount of usable information it gives, similar to how Wikipedia makes more errors than an encyclopedia, but makes up for it by being so convenient and covering such a vast array of topics.

Reading your Blueprint: DNA Fingerprinting

Who are you? That’s the question asked repeatedly in the song by The Who that CSI uses as it’s theme song. The answer, it seems, is found in the various pieces of evidence the Crime Scene Investigators find. Sometimes it’s pieces of fiber or pollen that eventually lead to the answer, but by far the most common method of determining identity is analyzing DNA evidence. What you see in the show is attractive people in lab coats taking samples of tissue, sticking them into some machine and then looking at a bunch of bars on a lit screen and saying things about what they mean. How is this any different from the old methods of reading patterns in tea leaves or goat entrails?

First off, I should probably point out that tv shows don’t always get things right. It usually takes much longer to get a result from a DNA sample, and if you recall the Casey Anthony trial and the OJ Simpson trial, DNA evidence does not always lead to a sure result. Well, what good is it, then? It’s not that DNA evidence isn’t useful, it’s just that there are circumstances where it isn’t available or isn’t clear. Similar to fingerprints.

So how do they analyze the samples they gather? They can’t use karyotyping usually, because they need living cells in order to do that. Thankfully, technicians don’t need to have DNA in chromosomes in order to be able to analyze it. As long as all they are looking for is straight DNA, they can get it from almost anything biological. Hair, skin, saliva, blood, and other fluids all contain viable DNA.  You still have to get it out of the sample first.

Close up of polymer gel structure. Water and small molecules can pass through spaces in the mesh. Image adapted from http://bit.ly/qvsOix

One way to do this is to crush and dissolve the sample using proteases. Proteases are enzymes that eat up proteins. They are present in a lot of different cells and even in some of the food we eat. If you’ve ever eaten a lot of pineapple and found your mouth sore afterwards, the reason is because of the proteases in the pineapple. This is also the reason why it is very difficult to make pineapple jello. Jello is a type of gel, a substance made up of proteins that form a mesh that traps water inside. If there is too much protease present, that mesh can’t form, and the Jello mix stays syrupy. The materials that keep your body’s cells from moving all over the place are also a mesh of proteins, and so if there is enough protease, some of this mesh will be dissolved. This is why pineapple can make your mouth sore. It is also why a strong protease can eat away the proteins that surround a DNA sample. Hair samples, in particular, have a lot of protein surrounding the DNA, so there needs to be a lot of protease to get to it.

Another way to get at the DNA is by using a detergent. Laundry detergents clean clothes by breaking up the oily materials that trap stains in the fabric. The membranes of cells are also made of oily material, so by using a detergent, we can wash away the cell membranes and get to the DNA inside.

Often both a protease and a detergent is used just to be sure the DNA is accessible. After this, the sample is put through a centrifuge to remove the lighter, non DNA molecules from the heavier DNA. Once the DNA is free, chemicals or heat is used to separate the two strands of DNA into single strands. This allows the restriction enzymes to work on the DNA. Restriction enzymes cut the DNA at specific areas that a technician can determine ahead of time. DNA is formed of four different bases: Adenine, Guanine, Cytosine, and Thymine, Each enzyme attaches to a certain sequence of these bases and cuts the DNA where ever that pattern shows up. DNA from different individuals will have different lengths between each recognized pattern and so will be cut up into differently sized sections after the restriction enzymes do their work.

Image from http://bit.ly/9h7sIW

The cut up DNA is then poured into a gel, usually made out of agarose or polyacrylamide. Just like Jello, these gels are made of long molecules connected together to form a mesh that partially traps water. The DNA fragments can travel through the spaces in the mesh, but not very easily. Lab workers apply an electric field to the gel using a tray with exposed electrodes that holds the gel immersed in a bath of mostly water mixed with some chemicals to help pass the electricity to the sample.  DNA is made of sugar molecules, and like most things made of sugar, it becomes negatively charged very easily.  In fact, unless DNA is in a solution with a high pH, it will have a slight negative charge, and so the electric field made by the tray device  pushes DNA down the gel toward the positive electrode in a process called electrophoresis.

http://www.biologyreference.com/Dn-Ep/Electrophoresis.html

The larger the piece of DNA, the harder it is for it to pass through the mesh of the gel. Smaller strands will therefore travel farther along the gel in a given period of time.Fragments of DNA that are the same size will be in the same area of the gel after they’ve been pulled through it, forming bands in those areas. These bands are still not visible, however, until the lab worker applies some kind of label to the DNA. This could be a chemical dye that binds to DNA, or DNA fragments with radioactive isotopes attached. The most common chemical dye is ethidium bromide, which is only visible under a black light. The lab worker can place a gel stained with ethidium bromide on a scanner attached to a black light to record the bands in the gel. In the case of radioactive isotopes, a film sensitive to x-rays can be placed on the gel which will turn dark where ever the radioactivity is.

Gels fall apart very easily, so many lab workers will transfer the DNA from the gel to a sheet of filter paper by pressing the paper to the gel and passing a current through it for some time. Then they can apply a label as before to see the bands of DNA.  These bands are what you usually see on tv shows where they compare DNA. By looking at where the bands from different samples show up, a lab worker can tell which samples are from the same source and which ones aren’t.

Debbie Knight at biologyze gives an excellent  detailed accounting of the process of gel electrophoresis here if you’re interested. She uses it to analyze proteins, but the idea is very similar.

Sometimes there isn’t enough DNA in a sample to get visible bands, in fact, this is usually the case. For these situations, there is PCR. PCR stands for Polymerase Chain Reaction. Polymerase is an enzyme that replicates DNA, but it’s more complicated than that. In order for the DNA to be replicated, it has has to be separated into single strands from its usual two stranded arrangement. This is called denaturing the DNA. In nature, this task is performed by proteins called helicases, however in the lab, heat or other chemicals can do the job.  Chemicals are somewhat difficult to work with, because they have to be washed out of a sample every time they are used. Heat is a much better denaturing agent, but most polymerase molecules can’t stand the heat needed to separated the DNA.

http://users.ugent.be/~avierstr/principles/pcr.html

Thankfully scientists found the molecule Taq, a DNA polymerase found in bacteria that can withstand the heat.  So what happens during PCR is that after Taq is added, the samples are first heated to a temperature high enough for the DNA to denature. Then the temperatures lowers, letting initiating sections of DNA called primers to attach or anneal to the fragments. Then the temperature raises again to a temperature that is ideal for Taq to replicate the DNA. Finally the samples are heated once more, so that the new DNA fragments separate again with twice as many fragments as before. This reaction is performed again and again, so that first two new strands are made, then four, then eight and so on, the amount of DNA increasing exponentially with every cycle. This way even with a miniscule amount of DNA you can have enough of a sample to work with.

 

Immunohistochemistry

Diagram of how an atomic force microscope works

So there’s this amazing protein you want to study, and it might very well change the world, but you have a problem: how do you study the protein when its too small for even a microscope to see?

There have been a number of solutions to this problem over the years, many of which are still being used. You could, for instance, study the protein’s effects by adding controlled amounts of it to a sample, or you could keep an organism from producing the protein  through surgery, drugs, or geneticsand see what what happens when it isn’t there. More directly, the obvious solution is to get a better microscope. What do I mean by a “better” microscope? Well, although there are some drawbacks, there are some microscopes that can give much higher resolution and/or provide added information about what you’re looking at.

One example of this is an atomic force microscope. How this works is that a small needle on a cantilever connected to a computer is dragged across what you want to look at. The computer then measures all the bumps that the needle encounters and gives a visual representation on the screen. As you might suspect this method has a number of drawbacks, (the cantilever is fragile, samples have to be specially prepared, etc) but it is possible to identify single atoms using this method.

Another type of microscope that can help you find a protein is an electron microscope. The idea here is to use electrons instead of photons to look at a sample. There are a number of drawbacks to this idea as well, for one thing, samples usually have to be “fixed” with a chemical such as Osmium tetroxide  and this can sometimes change the way things in a sample look. However, here the drawback can also be a good thing. What “fixed” means in this case, is that the molecules are attached to the things around them, and so aren’t going to move around all over the place like they would normally. If you want to see stuff moving around, you’re out of luck, but often you want to see where something is at a particular time and you don’t really want it to be free to wander.

There is another benefit to using electron microscopes. Parts of a sample will be darker depending on how electron dense they are after they’ve been fixed.  the electrons that the microscope uses to scan the sample will bounce off of that part of the sample, causing there to be a dark region. It isn’t always easy to tell which molecules will be more electron dense than others, but there are ways of making it easier. For one thing, if you have a molecule that you know is electron dense that you also know will form a bond with the protein you are looking for, you can add that to your sample and then you can compare the dark regions in the treated sample to how an untreated sample looks. The dark regions that appear in the treated sample but not in the untreated will most likely be your protein.

How confocal microscopy works

This trick also works with fluorescent and confocal microscopes. These microscopes shoot  beams of light at a constant, controlled frequency on the sample, causing some molecules to fluoresce, emitting light at a slightly different frequency. Confocal microscopes have the added benefit of being able to control where exactly the lasers focus so that it can show not only where something is in terms of up, down, left, and right, but also where it is in terms of depth. Once again, if you know a molecule is fluorescent and that it binds to the protein you’re looking for, you can add it to your sample and then check it out in the fluorescent microscope to find your protein. That’s great, but how do you find a fluorescent molecule to bind to your protein?

Diagram of antibody production

Well, your body has a ready-made system for finding molecules that will bind to proteins that has been tested over millions of years of trial and error. The immune system. If a virus or a bacterium enters your body and starts causing problems, your immune cells will start producing antibodies for the proteins that are present on the surface of the intruder, so that if it shows up again, it will be dealt with before it can cause any damage. Antibodies are large (by protein standards), y-shaped molecules produced by white blood cells. There are binding sites at the ends of the smaller arms of the y that bind to specific parts of a molecule. The binding sites act as a sort of lock, where the key is the part of the molecule the antibody binds to. There are a huge number of different binding sites that are available due to the genetic information encoding the antibodies getting shuffled and mutated all the time. When a cell in the body gets stressed, it sends out a signal that a white blood cell(a macrophage to be specific ) can respond to. This white blood cell then invites another cell (a T-cell) to take a look.  This T-cell will then go out and talk to another cell (a B-cell), which has a catalog of antibodies available for production that the T-cell can peruse by seeing if the peptide, or protein part, that’s in the stuff the macrophage ate, is also in the B-cell. If any antibodies from the B-cell  bind to something in the T-cell saw in the macrophage, then the B-cell knows to produce more of that kind of antibody. After that, wherever an antibody encounters its target, it triggers a response from other white blood cells.

Sometimes a cell might be stressed, but the antibodies will bind to something that isn’t the cause. Molecules, from peanuts, pet hair, pollen or just about anything might happen to be present in greater quantities than the thing that’s really causing the problem. This is how allergies happen.

That’s the bad news. The good news is that because antibodies can be found for almost any kind of molecule around, scientist can used lab animals to produce antibodies to the proteins they want to study. Furthermore, by manipulating the genetics of the animal, they can cause each antibody to be attached to a fluorescent molecule. The antibodies can be stored in a vial in a freezer and transported cold to labs all over the world. All a scientist needs to do then, is bathe whatever he or she is studying in a dilution of the antibodies, and then look at the sample with a fluorescent microscope. Wherever the sample fluoresces, that’s where the protein is.

This process is called immunohistochemistry. Immuno- because it deals with antibodies from an immune response, histo- from the Greek for tissue, and chemistry because it deals with the binding of molecules.

Antibody production diagram is from A Positron Named Priscilla: Scientific Discovery at the Frontier (1994) National Academy of Sciences (NAS) ( http://www.nap.edu/openbook.php?record_id=2110&page=69 ) all other images from wikipedia.

Myxobacteria are Awesome

Swarming myxobacteria
Image pulled from http://bit.ly/m51q1q

I am writing this post because myxobacteria are awesome, and I think more people should know about them.

You could go through your entire life without ever hearing about myxobacteria. Pretty much everything you need to know about microorganisms in general is that there are things moving around that are so small you can’t see them, sometimes they can help with things like digestion and pollution cleanup, and sometimes they can cause diseases such as salmonella, so you should really make sure to wash your hands, cook your food and  pasteurize things.

Everything beyond that is a detail that you can probably overlook without any serious repercussions.

But what’s the fun in that?

As with just about any facet of science, if you look into microbiology you soon find yourself falling through a magnificent and intimidating rabbit hole of information. It helps to have a specific thing to latch on to to make sense of everything.

Myxococcus fulvus fruiting body
image pulled from http://myxobacteria.ahc.umn.edu/whataremyxos2.html

Even focusing on myxobacteria gives you a lot to take in though. The picture above shows how they move around in swarms or wolf packs. The colors indicate the direction each cell is moving in. For instance, all the cells that are colored orange are traveling to the right. Why do they do this? How do they do this? They don’t have any flagella or cilia (not in the classic sense anyway) They release slime,( myxobacteria comes from the greek myxo for slime after all), but how does that help? I’ll say more about myxo movement in a later post, but there’s more.When food is scarce, myxobacteria start lumping together in visible fruiting bodies, about a millimeter tall,  like in the picture on the right. These fruiting bodies will eventually release bacterial cells with thicker cell walls that act as spores, eventually budding and making more myxobacteria, which then repeat the process.

Well, okay, that’s your basic fungus, right? But the weird thing here is that these are all still bacteria. They don’t even have nuclei! How do they show such complicated behavior?   How do they know enough to organize themselves into a heap to release spores? How is it that the spore cells look so different from the other cells?

Scientists know the answers to many of these questions, but others are still still a mystery. As you go through some of the explanations, you start to notice these strange correlations between how bacteria sense things and how social networks and mobs of people form and behave. You start to wonder about how some professions get more specialized and whether businesses and franchises are fruiting bodies. It’s eerie. And if these dumb bacteria act this way, is it possible that we act in similar ways because we’re in a similar environment? What can we take from that? Are we too much like bacteria, or not enough? What does this say about free will?

Deep, huh?

Like I said, Myxobacteria are awesome.