Tag Archives: PCR

How to Make a Mutant: Mutagens

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

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.