Tag Archives: DNA

Review of The Windup Girl

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Image from http://bit.ly/9zOK9t

It’s a dilemma we all face to one extent or another: we like technology, but we hate what it does to the environment. We like driving, but not oil spills. We like electricity but we don’t like to think about what ecosystems are being damaged to produce it. You’ve got solar cells? Great, what are they made of? Is that recyclable? We are in the process of resolving this conflict, but we’re not there yet. Let’s say the fossil fuels we rely on finally go out. Let’s say all the things environmentalists have been warning us about actually happen. What’s next? How would people cope?

The Windup Girl, by Paolo Bacigalupi, takes place in a different world. A world that is born after the world as we know it ends. The primary sources of energy are metal springs wound by hand or by the use of elephantine beasts of labor, and the methane produced when burning the refuse from men and beasts alike. The main police force is the Environment  Ministry,  who patrol the city in their white uniforms, ruthlessly burning or destroying anything that might pollute resources too much, or release plague into the populace. The only edible plants that survive are genetically modified to resist such plagues and even then have to be closely monitored. The “white shirts” are at constant odds with businesses, who often hire mercenaries to protect their cargo from destructions when bribes to corrupt white shirt officers don’t work. And then there are the people who are genetically modifying the crops. Called gene rippers, they are loathed by all because they are the source of the plagues that threaten the populace, but tolerated because without them, there would be nothing to eat.

From this short description, you can already get an idea of the vast amount of world building that Bacigalupi did for this book, and his characters are as complex as the world they inhabit.

Anderson Lake is a gene-ripper who has a cover job as the overseer of a massive kink spring factory. The factory is huge, with giant elephant beast turning giant cranks in giant baths of algae. Helping him out with the logistics of this operation, and with bribing the necessary officials is Hock Seng (pronounced hock sahn), an Chinese refugee from the genocidal massacres that had taken place in Malaysia several years before. Hock Seng’s entire family was killed during the tumult there , and he had barely made it out alive. So now, even as he pretends to do Anderson’s bidding, he is secretly making plans to steal enough money to establish himself as a merchant in a country where he won’t be persecuted.

The book starts as Lake finds a bizarre fruit in a market that seems to be immune to plague. Realizing that this means there must be another Gene-ripper around, and that this gene-ripper must have access to other sources of genetic information, Lake quickly makes meetings with important business leaders in order to leverage himself into getting access to the gene pool. One of these meetings takes place in a brothel where a beautiful looking Japanese girl, with skin eerily white and smooth, serves Lake. She moves in stops and starts, identifying her as a genetically modified or “new” person. She is Emiko, the wind-up girl.  She is lower than a slave in the brothel, only allowed to exist because of the bribes paid to white shirts. She is mocked, ridiculed and despised by almost everyone she comes into contact with. But Lake is intrigued by her, and he tells Emiko of a village of wind-ups to the North where Emiko might be accepted. This gives Emiko hope for the first time in years.

Finally there are Jaidee and Kanya. Jaidee is the captain of a squadron of white shirts. He started out as a Muay Thai boxing champion and carries his fighting spirit into his job. When there is a ship full of suspicious cargo, he doesn’t bother trying to sort through it, he burns it all. Even while most of the Environment Ministry are despised by the people for their corruption and meddling, Jaidee is well-liked because of his pure motives. But his exuberance has cost a lot of powerful businessmen, and they are going to try to make him pay for it.

Kanya is Jaidee’s first officer, and where Jaidee is boisterous, Kanya is quiet. She rarely ever smiles. She seems at first to be a relatively minor character, but she has many secrets, and after a series of catastrophes, she becomes one of the most important characters in the book.

The Windup Girl is science fiction written as epic fantasy. If you’re ready for it, the plot is intricate and engrossing, but if you aren’t, it can also be complicated and confusing.  There are also several sections depicting gory scenes, and there are two rape scenes that I find disturbing. These scenes aren’t gratuitous. They are important to show the arcs of the characters, but you should know this isn’t a book of chaste kisses on gleaming spacecraft or anything. This is a gritty depiction of an all too possible future, a future that you could argue is already taking place in some developing countries.

So why should you read it if it’s so depressing? First off, I wouldn’t call it depressing. I would say illuminating and even uplifting to an extent. The book illustrates an important point about the conflict between technology and nature: there is no real conflict. Technology comes from us, and we are part of nature. Nature changes all the time, and like all creatures, we must adapt or perish. We can now control larger and larger areas of nature. As part of nature, we have to adjust to this. We can’t eliminate technology, but we can’t be reckless with it either. We’re grabbing the steering wheel of the Earth-mobile. If we don’t pay attention, this could go very badly.

This isn’t the only theme of the book,  and I’m not sure if the author would even agree completely with my interpretation. You don’t have to agree with the theme to like the book, though. The characters carry the story. They are all flawed people trying to do the right thing even while they end up fighting against one another. Anderson Lake is my least favorite of the point of view characters, but even though he can be arrogant and inconsiderate, even cruel, he has a discernible arc, and his motives are understandable.  All of the characters, Anderson included, had numerous moments where I was rooting for them.

http://paolobacigalupi.blogspot.com/

On the negative side, there were some ends that were a bit too loose at the end of the book. Particularly for Hock Seng. He was the biggest underdog in the story and his fate was a bit too unclear for my taste. Although some things made sense after thinking about them for a while, the ending initially felt a little too abrupt too. I wasn’t sure about the arc of all the characters. Once I figured out how everything tied togethera couple days after finishing the book, I was struck at how moving it all was. As I figured out, there is an emotional theme along with the semi-political one. To paraphrase Jaidee…Cities don’t matter. Plans don’t matter. In the end, what matters is people.

There were some moments as I was reading to the book that I didn’t like it much at all, mostly because some of the scenes with Emiko were a bit hard to get through, and because it took a while to get a grasp on the plot, but by the end of the book, it was a 7/10, and after I reflected on it, it reached 8/10. (This is a pretty high score. For comparison, the Lord of the Rings movie series gets an 8/10 from me).  I bought the book after attending a panel at The Southern Festival of books where Bacigalupi was a guest. He does an incredible amount of research for his books and seems to look deeper into things than most people. After reading this book, I want to meet him again so I can be properly impressed.

Gels

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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

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(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.

How to Make a Mutant: Mutagens

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http://bit.ly/p2fCgl

The term “mutant” gets bandied about quite a bit in popular culture. It can mean some freakishly deformed animal or person, or, more recently someone with magical, almost god-like powers (if you think that the X-men stories are completely scientifically accurate, then you have a disturbingly unrealistic understanding of the universe). But the reality is that mutants are all around us. The word comes from the Latin mutantem which means to change. In some sense, then, we are all mutants, as our genes are naturally a mix between the genes of our parents and therefore are always changing. Usually though, by “mutant” we mean some organism that has had their DNA altered by artificial means. Even this more focused definition still applies to an astounding number of the things we look at every day.

There are basically two ways of making a mutant: using a mutagen, or specifically targeting  an organism’s genome. We’ll talk about mutagens first. A mutagen can be anything from nuclear radiation to insecticide. Anything that’s a labeled carcinogen is also mutagen, as cancer is perhaps the most common form of mutation. Since cancer happens naturally, it’s perhaps a little  incorrect to put  it under the heading of “mutation,” but as some unfortunate people are intimately aware, cancer can be caused by man made materials, so it does fit.

Before we get too ahead of ourselves, we should first go over how a mutagen might make a mutant. The way it works is this: every living thing starts out as a single cell. How this cell behaves is determined by the genetic information in the DNA of the cell. This behavior includes how it divides, what structures it makes, whether it moves around, how it connects to other cells, you name it. If this cell is exposed to a mutagen, then this DNA might be altered. For instance, if the cell is subjected to ionizing radiation,  subatomic particles will scour through all the material of the cell including DNA, knocking electrons and possibly even whole atoms off the molecules that make up each material. If the intensity of the radiation is low enough, most cells can repair this damage, but some damage might be too severe to repair, or might be missed by the repairing processes, and if this happens to DNA, a mutant can result.

Ionizing radiation, as you might imagine, is a rather ruthless mutagen. It’s a bit like trying to hit the bull’s-eye of a target with a shotgun. You might get lucky and hit the area you want, but even in the best case scenario you’re going to have collateral damage. So while this type of mutagen is the most commonly found in nature, it isn’t something you want to use to find a beneficial mutation.

A slightly more nuanced approach is to inject material that looks like a section of DNA into the originating cell while it’s dividing. This approach only affects the DNA, and so you don’t have to worry about killing the cell outright, but it’s still random and so it could cause changes that an organism won’t be able to survive. There is a more sophisticated version of this involving actual foreign DNA, but we’ll get to that in the next post.

One of the mutagens most commonly used in labs is a chemical called EMS (Ethyl MethaneSulfonate). This chemical will affect the DNA of a cell by affecting  only a single nucleotide base pair. This allows much more of the mutants to divide and develop fully into adult organisms.

All these methods will produce mutants, and this is far from a complete list, but scientists usually have a specific mutant in mind. For example, a scientist might be interested in muscle growth and wants to find an animal mutant that will mimic a known human disease.

http://bit.ly/pLanTO

The first step for this is to find the right animal model. Animal models are used in many kinds of research genetic and otherwise. A human model wears fashions or tries different products so that we can see how they supposed look or work in an idealized environment. In a similar way, an animal model is given different ailments so that scientists can see how the animal responds to different treatments and other situations, with the hope that a human might have the same result. Humans are not all that different from other animals, as even Aristotle was aware, (We should venture on the study of every kind of animal without distaste; for each and all will reveal to us something natural and something beautiful. -Parts of Animals I.645a21)  but certain animals are better for studying different systems. If you want to study the higher functions of the brain, for instance, you probably want to work with mice, or chimpanzees. If you are interested in the steps involved with development from a single cell to an embryo, however, you might use zebrafish, as zebrafish embryos are transparent, allowing you to see many processes hidden in other animals.

One thing that is common for most animal models is that they tend to have faster life cycles, and produce more young. If you are a graduate student hoping to get your PhD in two years, you don’t want to have to wait ten months for an animal to get born, just to find out it doesn’t have the right genetic make up and you have to start all over again. In lectures, scientists often talk about how expensive each animal is. In other words, how much grant money goes into studying each individual animal. If a study is supposed to follow the entire lifespan of a rat, then it’s going to take four to seven years, and so that rat is going to be very expensive. If a scientist does a similar study with a fly, on the other hand, it will only take a few months and therefore be only a fraction of the cost. Also, while there maybe eight to twelve rats born from a mother, which is a good number in comparison to a chimpanzee, there still may not be enough chances for the pups (baby rats) to have the right genetic make up. If one of the pups needed for the study dies, it can be devastating, while if a fruit fly dies, there might be forty other flies to take its place. This is one reason why fruit flies are used often in genetic studies.

Two fruitflies contributing to research -http://bit.ly/oy62XW

Let’s look into how to make a fruit fly mutant. The procedure is typically to subject fertile, male flies to a mutagen (EMS for example) for a period of time, and then allow them to reproduce with normal female flies. Some degree of care must be taken to ensure that the female flies are virgins, so that there isn’t any chance of another fly’s genetic material getting involved. Thankfully virgin females are paler and a black dot is visible on their abdomens. They are therefore distinguishable from older females, which don’t have the black dot, and males, which have darker coloring and a reddish structure at the ends of their abdomens. To examine these features, a researcher can take flies and subject them to CO2 gas, which knocks them out. They can then manipulate them using tweezers and a low magnification microscope. The virgin female flies are sequestered in a separate vial with a mutagen-treated male and allowed to mate. The female will lay mutant eggs, which will eventually become mutant larvae, and then mutant flies. Depending on what trait a researcher is looking for, they will analyze either the larvae or the flies for altered behavior or health.

Wild banana -http://bit.ly/l1jut6

This method  of producing mutants has been around for decades. You might think that they are science fiction things, but if you walk into any sophisticated biology lab and talk to somebody there, you’ll find that mutants are not only studied a lot, but they are almost taken for granted.  Furthermore if you take away the use of mutagen, this kind of directed evolution has been around for ages.Without human intervention bananas are, fat, green, cumbersome things that are difficult to work with. By cultivating the trees that produced the tastiest, easiest to eat fruits, however, humans managed to breed the trees to produce the banana we know and love today, a fruit that fits so nicely in the hand, and opens so easily it seems like it was designed for us. It seems that way because we designed it.

This also has happened with animals. Geneticist Dmitri Belyaev managed to domesticate foxes, by breeding the ones that were the most tame. The breeding program was successful, but oddly the tame foxes began to look an awful lot like dogs.  Belyaev’s research suggests that many of the same genes that control physical attributes, also control behavioral ones. Just as he domesticated foxes within his life time, wolves must have at one time been domesticated by early humans. In other words, when you look at a dog, you’re probably looking at a mutant wolf.

Reading your Blueprint: Genome Sequencing

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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

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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.

 

Reading Your Blueprint: Karyotyping

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DNA is  what makes us who we are. It identifies us. The government uses it to control us. Well, that last one isn’t quite true…yet. Still most of us, even those of us who should know better, treat DNA as if it were magical fairy dust that wizards in lab coats use to answer questions. But the only difference between the wizards and you is some knowledge and equipment. This blog will help with the knowledge part.

There are three main ways to use DNA to tell something about an individual: Karyotyping, DNA fingerprinting, and Genome sequencing. I’ll go over Karyotyping in this post and tackle the other methods later.  Karyotyping is basically looking at the chromosomes of a cell as it’s dividing. Cells divide to make new cells, but in order to make sure each new cell has everything it needs, everything has to be copied, including the genetic information. Unless the cell is a bacterium (or an archeum), it will keep its genetic information in a sort of ball of denser material called a nucleus. Most of the time this ball is all we see of the cell’s DNA, but if we add a dye and watch the cell divide, we can see several strange, threadlike structures, or bodies, that are colored by the dye. These “colored bodies” are the chromosomes, named after the Greek “chromo” for colored and “soma” for body. A chromosome is actually DNA wound up around proteins and then wound up again and again until it becomes a tight tangled mess, similar to what happens if you twist a coiled telephone cord.

As you look at  chromosomes under a microscope, in other words, you are actually looking almost directly at DNA.  Normally the chromosomes are haphazardly arranged around the nucleus of the cell, but after taking a picture of them, you can rearrange them so that they’re aligned the same way and in order from largest to smallest. This is a Karyotype.

Usually a lab worker who wants to karyotype a person’s blood, will look at some white blood cells, which are easier to work with for a variety of reasons, and wait for themto start dividing. Then, he or she will use a drug such as colchicine or vinblastin to stop the cell from dividing completely. This allows the technician to look at all the chromosomes under the microscope and analyze them using dyes that bind to different genes. The lab worker or another scientist or technician can then compare the karyotype of one individual to another to see what differences they are in where the dye shows up.

The drugs used to stop the cells from dividing have interesting histories. Colchicine started out as the active ingredient in an herbal remedy extracted from autumn crocuses to treat gout and inflammatory arthritis. Crocus extract was used as such as far back as 1500 BCE though it was only isolated from crocus extract in the late 1800’s. Colchicine is still used to treat severe cases of gout today, however, the difference between an effective dose and a toxic one is rather small, so takers of the drug have to be careful.

Chromosomes of a cell. Vinblastin or colchicine used to keep them from separating.

Vinblastin also comes from a plant extract, this time from the madagascar periwinkle plant. The plant was originally crushed into a tea, and researchers noted that people who drank the tea had a lower white blood cell count, leading researchers to look into the active ingredient as a possible treatment for diseases that affect white blood cells.

White blood cells are primarily responsible for the body’s immune response, which causes inflammation, the primary complaint of gout and arthritis sufferers. These cells also  affect a number of diseases, including cancer. White blood cells use microtubules to initiate movement, and all cells use microtubules to separate chromosomes as they divide. Cochicine and vinblastin both inhibit the production of microtubules, which is why the drugs are useful in karyotyping as well as a number of other diseases.

The easiest thing you can do with karyotyping, after determining species, is determining sex. A normal human being has 23 pairs of chromosomes and sex is determined from the 23rd pair. Genetic females have two longer chromosomes, called “X” chromosomes here, while for genetic males one of the chromosomes is shorter and called a “Y” chromosome. Sometimes a person might have two or more X chromosomes along with a Y chromosome in a condition called Klinefelter’s syndrome. There are also a number of other possibilities, such as XXX or even XXXY. All these scenarios usually result in learning disabilities, and regardless of how many X chromosomes there are, if there is a Y chromosome the person will be physically male. These situations where there are more than two sex chromosomes are examples of aneuploidy.

Karyotype of a normal male. From biology.iupui.edu

The word “aneuploid” comes from four Greek words mashed together: “an-” for not, “eu” for good,”ploos” for fold, and “oidis,” which means form or type. So all together you have “not good fold type,” a set of chromosomes that is not correctly paired. This sort of situation can occur in other chromosomes as well, such as in the case of Down syndrome, the second most common inherited  form of mental retardation, where there is an extra copy of chromosome 21.

In biological terms, “-ploid” refers to how many complete sets of chromosomes there are. The normal number of copies for a set of chromosomes is two, so most cells are called diploid. Sperm and eggs only have half the normal amount of chromosomes, so they are haploid. Plants and some other organisms can have more than two sets of chromosomes, making them polyploid. While polyploidy can occur in human fetuses, it never results in birth. In fact no vertebrate animal can be polyploid.

A normal x chromosome on left, fragile x on right.

Fragile X is the most common inherited form of mental retardation, and while it is not a aneuploidy disorder, diagnosticians can also identify it using a karyotype. In fragile X one of the x chromosomes looks thinner in an area that has a long string of the same DNA sequence. Because of this repeated sequence, a protein required for normal development can’t be produced properly and the neurons of the brain cannot form the proper connections as a result.

Diagnosticians can tell that someone has Fragile X from the way a certain area of a chromosome is affected naturally, however they can also tell other genetic attributes by how the chromosome is affected by the dye they use. Giemsa, the most common dye used for karyotyping, will concentrate in different bands on the chromosome, which you can see in the picture of the normal male karyotype above. By comparing where these bands show up between different karyotypes, researchers can begin to find abnormalities and differences that may have something to do with how a person looks, acts, or feels.

Other labeling techniques have also been used to get more information from chromosomes. Especially exciting is the work done on telomeres, which can be labeled using a protein attached to a fluorescent marker. These are the ends of the chromosomes, which are made up of repeating patterns of DNA that act like the aglets of shoelaces, keeping the chromosome from shortening prematurely. Telomeres have been found to be  linked with the aging process. Although the telomeres will shorten and lengthen throughout life, various stresses can cause them to shorten more than usual. It seems the older we are, the shorter our telomeres get. If we can figure out how to lessen the shortening process, we may find a cure for aging itself.

TELOMERES cap the ends of chromosomes. Image: WIKIMEDIA COMMONS/NATIONAL HUMAN GENOME RESEARCH (user GIAC38)

Suggestions? Corrections? Questions? Observations? I’m trying to cover a lot of ground here without letting things get too complicated, so I’m bound to make some mistakes. Please feel free to comment on this post or email me at zorknot (at) gmail.com about what I’ve written.