The AP Biology exam for 2017 is set for the morning of Monday May 8th. So it’s that time of the year again to begin reviewing past concepts and doing practice exams.
The free response questions on the past four exams didn’t touch so much on a very important concept, the tools and techniques of biotechnology. So, in this post we’ll review some of the major tools and techniques while going over Question 4 from the 2007 AP Biology Exam. On average students scored 2.52/10 points. This post will show students how they could have maximized their score and give a review of important terms.
A bacterial plasmid is 100kb in length. The plasmid DNA was digested to completion with two restriction enzymes in three separate treatments: EcoRI, HaeIII, and EcoRI + HaeIII (double digest). The fragments were then separated with electrophoresis, as shown.
a) Using the circle provided, construct a labeled diagram of the restriction map of the plasmid. Explain how you developed your map.
b) Describe how:
- Recombinant DNA technology could be used to insert a gene of interest into a bacterium
- Recombinant bacteria could be identified
- Expression of the gene of interest could be ensured
c) Discuss how a specific genetically modified organism might provide a benefit for humans and at the same time pose a threat to a population or ecosystem.
In order to answer this question you first had to know what they were asking for, so let’s review some terms.
A plasmid is a circular double-stranded DNA molecule usually found in bacteria that is capable of replicating independently of the cell’s chromosomal DNA. Usually plasmids allow the bacteria to develop some advantage such as antibiotic resistance. A bacterial cell can have more than one plasmid, will express the genes on those plasmids and will replicate each plasmid when it divides. In this way each daughter cell will get a copy of the plasmid. Plasmids are often used for cloning purposes since genes of interest (fragments of DNA) can be inserted into plasmids and introduced into the bacteria by bacterial transformation. In bacterial transformation, competent bacteria where the cell wall has been made permeable to genetic material can take up a foreign plasmid. The bacteria that have taken up the plasmid can be selected for usually by growing the bacteria in a certain antibiotic to which the plasmid DNA allows resistance. In this way, as the bacteria divide the plasmid and thus the genes on them will be amplified.
These are enzymes that cut DNA at specific recognition sites that are usually 4 to 8 base pairs in length. The sites are usually also palindromic, meaning they read the same forwards and backwards. The restriction enzymes will also produce “sticky” or “blunt” ends. These ends can be used to insert the gene of interest into a plasmid by ligation.
Ex. Sticky end where bases are left unpaired after a cut
Ex. Blunt end where all bases are paired after a cut
Gel electrophoresis separates molecules of DNA on the basis of their rate of movement through an agarose gel in an electric field. Remember that DNA is negatively charged due to its phosphate backbone and will flow from cathode (-) to anode (+). Smaller fragments of DNA will travel faster and thus be farther away from the starting wells. Similarly, proteins can also be separated using electrophoresis.
Using restriction enzymes and gel electrophoresis the restriction sites on a segment of DNA, usually a plasmid, can be mapped. This is useful when trying to identify whether a gene of interest was correctly inserted into the plasmid.
Part a) Using the circle provided you were to draw and label a restriction map of the resulting gel electrophoresis as well as explain how you came up with the map. The restriction enzyme EcoRI resulted in two bands of DNA, one that was 70kb and another that was 30kb. The whole plasmid is 100kb so from this information you can deduce that there are two sites where EcoRI cuts the DNA, since 70 + 30 = 100. So place two EcoRI sites on your circle.
Now we look at the fragments that are created by HaeIII. A 60kb and a 40kb fragment which equals 100kb as well. So we know there are only two sites where HaeIII cuts as well. Hold off on putting the HaeIII sites. The last restriction digest was a double digest using both EcoRI and HaeIII. Since both EcoRI and HaeIII result is two cuts each using a double digest should result in 4 fragments. In this case those fragments are 40, 30, 20, and 10kb. Using trial and error place the HaeIII cut sites so that the distance between the EcoRI sites doesn’t change and so that you get four cuts of the sizes 40, 30, 20, and 10kb. I would start by splitting the 70kb into two cuts one of 40kb and one of 30kb. And split the 30kb into one 20kb and one 10kb as shown below.
However, you aren’t done. You need to make sure that the HaeIII sites are 60kb from each other one way and 40kb the other. In our map above this is not the case so you should rearrange the numbers.
The resulting map should look something like this. Where EcoRI cuts at two sites that result in 70kb and 30kb fragment and where HaeIII results in a 60kb and 40kb fragment. In your explanation you should describe the process of trial and error that you used to come to this conclusion. DO NOT waste time providing information that was not asked for as this wastes valuable time. So there is no need to explain gel electrophoresis!
Part b) This section asks you to describe how a gene of interest is inserted into a bacterium, how recombinant bacteria (bacterially that have been genetically modified) can be identified and how expression of that gene of interest can be ensured. First, the gene of interest needs to be cut from the source, which can be accomplished using a restriction enzyme. This same restriction enzyme should be used to cut the plasmid that the gene will be inserted into to ensure compatible ends. The gene of interest and plasmid then need to be combined so that the complementary ends can match up and join together. Once the gene of interest is inserted into the plasmid, this plasmid needs to be incubated with competent cells so that these cells can take this plasmid up. To identify whether the bacteria has taken up the plasmid the bacteria can be grown on antibiotic plates as long as the plasmid has a gene for antibiotic resistance. To ensure that the gene of interest is being expressed the gene of interest can be inserted downstream of a promoter that can be induced when giving certain nutrients such as lactose. Remember that the promoter is the region on DNA that initiates transcription. Make sure you understand the process of gene cloning because it’s bound to come up on the exam! Here is a link to a great overview.
Part c) Genetically modified organisms are the results of genes being artificially inserted into the genome of organism. They are used all across science to produce new forms of drugs, vaccines, and even to produce food. Bacteria have been modified to produce insulin to help patients with diabetes, model organisms are genetically modified in research settings to test the affects of a certain gene product, and crops can be modified to produce a certain color, smell, or prevent against plant disease. Genetically modified organisms provide a benefit for humans in many ways especially when it comes to growing large amounts of food. Crops can be modified to become resistant to certain bacteria that threaten the yield thus increasing the amount of food available for consumption. At the same time this poses a risk because the bacteria may become resistant to the antibiotic the crop is producing and cause harm in the long term. For this part it was important to be as specific as possible and describe your ideas in a scientific manner.
The AP biology exam is meant to be challenging but knowing how commonly used tools and methods are used is essential! Make sure to answer each part of the question completely and label all your answers to maximize your score!
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In shows like CSI, Miami, New York or wherever they often throw up the term DNA fingerprinting. One of the most common methods of DNA fingerprinting is something called PCR and what it's all about is using PCR and Gel Electrophoresis to examine DNA that's what they mean by DNA fingerprinting it's not really somebody's finger print. Now PCR stands for Polymerase Chain Reaction which is a process for copying DNA and what it does is that it uses a special heat stable DNA Polymerase to copy a specific gene that you're interested in.
Now Gel Electrophoresis is this idea of using the fact that DNA is negatively charged to take your copy DNA put it into the agarose gel or some other materials and then you use electricity to drive that charged DNA through the gel and because that gel acts like an obstacle force it separates up the DNA fragments based on their size. Let's take a closer look at this YouTube video that shows the process known as PCR and we're inside of a test-tube filled with DNA from suspect if we're in CSI but all we're interested in, is this one particular section of DNA called the target sequence highlighted in green. Now this is going to take advantage of some of the steps involved in DNA replication the process of copying DNA.
Now normally with DNA replication we have to open up the helix, well to open that up in your cells you use an enzyme. In this test tube we're going to heat it up to 95 degrees Celsius which will separate the two sides, because that is almost boiling temperature. Now we cool it a little bit and allow a premade thing called a primer that tells which gene we're interested in copying to come in and so by cooling to right around 60 odd degrees or so that allows the primer to bind to our target sequence. Now the orange little things is that enzyme that can survive these high temperatures. And these green guys with sticks on them, those are the nucleotides the raw material for building our DNA copy. So the enzyme does what it's supposed to do, it finds the primer and says okay and it starts copying, and it keeps going.
And if you give it enough time it'll finish copying the entire molecule going this way and that one will copy going that way. Remember the two strands of DNA are anti-parallel, they go on opposite directions. But we only give it maybe 2 minutes at most and so at that point we then let it stop and we're at the end of cycle one. And so now that we're done with cycle one we can begin cycle two and it's the exact same thing, we heat it up to 95 degrees Celsius which is enough to separate our old, original template strands and our newly made copies. We cool it to 60 degrees Celsius that's cool enough for the primers that still are floating around in the test tube to bind to the beginning and end portions of our gene of interest. Then we go to the right temperature for the enzyme, the DNA polymerase it finds the primer and goes okay and it starts to copy and that's the end of cycle two.
At this point we have 4 copies now each of our copies contains information that's not part of our DNA but at the beginning of cycle 3 when we heat it up notice there's a couple of short little segments that are only the length of our gene of interest. We cool it, primer stick, the tag-primers comes along and binds it, it's called tag-primers that's short for thermokineses which is just the name of the creature came from but now we have a couple of our target molecules made. So we're ready to begin I believe this is cycle 4, so again we're going to heat it up, that' the end of cycle 3 so we heat it up for cycle 4, we separate our strands, we cool it enough for the primers to come on in, they bind to the beginning and end portions of our DNA gene tag polymerase does it's copying job and again we've made a number of copies of just the size that we want. Now original we had more of these longer ones but now we're starting to get more and more of the shorter ones.
As we begin cycle 5 we do the exact same thing over and over that's why it's called a chain reaction, each time we're doubling the number of our copies. And we just run it through, and this is such a simple process, this is one of the reasons why this when it was first invented people were going wow how did they think of this and there's a lot of a pack full of stories about how the guy actually did think of that, but he is niow a very rich man because everybody does this process. Now you can see we've got 22 molecules and that's after only 5 cycles. Each cycle takes maybe 90 seconds to couple of minutes, so you this 30 times and that takes you maybe 90 minutes and at the end of it you've got a large number, billions of copies of your target.
Now you can't see an individual molecule but you can see billions of molecules. Now how are we going to visualize this? How are we going to see how big that is? That's where the Gel Electrophoresis comes in. So we'll stop the YouTube and we'll go to a PowerPoint slide and let's imagine we've done a DNA fingerprint of 3 people and we're looking to see, we're not using this to identify who they are like you would see a side, but we're doing this trying to figure out what genes do they have? Let's suppose we've found a gene that if you have a longer version of it, you're more likely to get a particular cancer. If you have a short version of it, you're less likely to get a particular cancer.
Well we have patient 1, patient 2 and patient 3, now in this fourth row here what we have is pre made DNA so that we can use it like a ruler and what we do is we loaded our DNA samples into these holes here called the wells. We turn on the current, this end is negatively charged, this end is positively charged DNA has a negative charge to it so it is repelled by the negative side and goes zoom towards the positive end. And little guys one thousand base pairs long move a lot faster than the big 10,000 base pair of long pieces of DNA. Now this person here, we only see one band, this person here we see one band, this person we see two. Why is that? Oh yeah everybody has two copies of every gene, this person has two copies of the long version. Their homozygous for this particular condition.
This person here is homozygous for not, for the shorter version i.e. not having the cancer. This person here is heterozygous, so this person has a medium chance, this person has a low chance, this person with a double dose of the longer more, greater chance of having cancer gene. This person is at greater risk, and this is one of the uses of DNA fingerprinting is to assess your genetic risks so that you can make wise, intelligent decisions about what you're going to do in your environment. If this was you I would not smoke and watch your diet and do everything else you can to keep this genetic prediction from becoming a genetic reality.