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Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
When people think of DNA, they often first jump to unrealistic depictions of DNA's use in crime scenes from television shows and movies. There is a lot wrong with how the science is shown in those programs, but there is one piece that is true.
A very small amount of DNA can prove crucial in figuring out a culprit (or when analyzing DNA for less interesting reasons).
DNA is tiny - we all know that. But in order to run lots of tests on a sample, it is important that we can create copies of that DNA. That way we won't ruin all of the sample!
This is where polymerase chain reaction (PCR) comes in. It is essentially a laboratory version of DNA replication. We will not get into the specifics of the process for this course, but just know that PCR allows us to take one copy of DNA and quickly copy it as many times as we would like to make a bigger sample that is easier to use.
Restriction Enzymes
Restriction Enzymes
Now that we can copy lots of DNA, that means we can mess with our samples in the lab to analyze them. One of the most important things that scientists will do is chop up the DNA with restriction enzymes. These are natural enzymes made by bacteria (and other prokaryotes) that will break the two strands of DNA.
Remember, these are enzymes, so they do not just cut wherever they feel like it. Enzymes are very specific to their substrate. So these enzymes will bind only to specific sequences of DNA. Each restriction enzyme has its own unique substrate. As a result, cutting up DNA by putting it into a solution with restriction enzyme A will result in different pieces than one placed in a solution with restriction enzyme B.
Here is an example of the way in which one restriction enzyme may cut up DNA. It will locate the sequence shown, and then chop up the DNA along the blue line. It will then move on and try to find that same sequence again.
Often, the same sequence can appear many times along the same DNA strand. Remember, all individuals have unique DNA sequences, which determine their alleles. This means that chopping up my DNA with restriction enzyme A may result in 30 pieces, but chopping up someone else's DNA with the same restriction enzyme may result in only 5 pieces depending on how often that sequence occurs in our genomes.
Gel Electrophoresis
Gel Electrophoresis
Once DNA has been chopped up in the lab, it can be placed into a gel. DNA samples (filled with all those fragments) are pipetted into the sample wells shown here. The gel has little pores, or holes, in it that DNA can make its way through.
DNA is actually negatively charged, so it is attracted to positive charges and repelled by other negative charges. In gel electrophoresis, a power supply is connected to the gel so that there is a positive side and a negative side.
Notice that the DNA begins near the negative side, so it will 'want' to make its way over to the far side.
DNA can make its way toward the positive side through those pores in the gel. It's not easy to get through this way, so some pieces will not make it far.
The smallest fragments of DNA will make it the furthest because it is easiest for them to maneuver through the pores. The large fragments of DNA will not make it as far, so will be closer to the negative side than the short ones.
Often you will see an image of a gel electrophoresis experiment like that shown here. You should be able to recognize where the larger and smaller fragments end up.
In reality, these gels can look something like this image (often even blurrier than this depending on the sample and your lab equipment!). As a result, most diagrams you see for AP Biology will be simplified versions like those above and in the following section.
DNA Fingerprinting
DNA Fingerprinting
More interestingly, however, this gel pattern actually will allow us to analyze individuals because everyone's DNA is unique, acting like a genetic fingerprint.
As long as the same restriction enzymes are used for each sample, unique DNA samples will lead to unique band patterns.
As a result, a DNA sample found at a crime scene, for instance, can be analyzed and compared to DNA samples from given suspects. The suspect whose gel bands match the sample would be the individual from whom the sample came.
In this particular image, you can see that Suspect 2's bands match the sample because their DNA fragments were the same lengths after being exposed to the restriction enzymes.
This same methodology can be applied to maternity and paternity testing as well. Remember that these bands are made of DNA fragments, and DNA comes from one's parents. So half of your DNA came from your biological mother and the other half from your biological father.
So you should see bands in common with both parents. All of a child's bands should be accounted for by looking at both parents. Think of it this way: a child has 4 DNA bands. They had to get them from somewhere - their parents, of course. So we should find those bands in their parent samples.
If we compare the mother and child DNA in this example, we see that they share two DNA fragments (highlighted in red). However, the child's other two strands do not match the mother, and therefore must have come from the other parent, the father. In order to determine who is the father, we just need to compare the DNA bands of the different men to those bands that are unaccounted for.
We can see some bands in common with both men 2 and 3 (highlighted in blue). However, man 2 is the only one individual who could have supplied all the DNA from the child that did not come from the mother. Therefore, man 2 is the father.
Plasmids & Conjugation
Plasmids & Conjugation
In order to understand some of the most recent developments in genetics, we will have to review a little information about bacteria. Recall that bacteria have circular chromosomes (see the blue DNA here).
However, bacteria also can have plasmids, which are also circular DNA, but they can be expressed and replicate independently from the rest of the DNA.
These plasmids allow bacteria to share DNA with one another. They can transfer plasmids from one bacterium to another via conjugation (shown here).
Essentially, this allows bacteria to share traits with one another. This would be like a friend of yours sharing their eye color because you liked it. Now you have their eye color.
Do not memorize any of this process.
Created with BioRender.com
With contemporary biotechnology, we can essentially form plasmids that carry traits we want bacteria to gain. Then we can introduce those plasmids to a population of bacteria, and they will take in these traits. This will cause those genes to be added to the genome of the bacteria, to be passed on in future generations. Obviously this is an oversimplification, but this is essentially all you need to be familiar with for AP Biology.
Genetic Engineering with CRISPR-Cas9
Genetic Engineering with CRISPR-Cas9
Similarly, our ability to manipulate the genomes of eukaryotes has also improved with time. We have begun to use CRISPR-Cas9, a DNA-editing structure naturally formed in the immume response of bacteria, to edit the genomes of cells in laboratories. Cas9 is the name given to the enzyme, but there is also some RNA to guide it.
Essentially, CRISPR edits genes by cutting DNA very precisely and inserting or changing DNA. Then, the normal DNA repair processes that occur in the cell take place, incorporating the new (or changed) DNA into the chromosome. Again, this is an oversimplification, but more than enough for this course.
This technology has all come about very quickly, obviously. Remember, we didn't know what DNA's structure was until 1953! So as a society, we will have to tackle some very complex issues with this technology...
Will be start making designer babies, allowing you to choose traits in your children? Will we be bale to cure genetic diseases?
These may sound like simple issues, but the precedents set now will have monumental effects on what is allowed in the future.
We need to tackle these questions now because gene editing is already here. In fact, He Jiankui (shown here), a scientist in China, announced in 2018 that he had created the first genetically edited babies in twins Lulu and Nana.
This is the first confirmed case of editing the germline in humans, essentially making it so that any changes could be passed on to the individual's offspring.
Since the announcement, he was condemned by many scientists for lack of following safety protocols and precaution. He was, along with collaborators, found guilty of illegal medical practices. He was sentenced to three years in prison.
The technology is here and it has amazing potential. How that potential is used is going to be up to science and society.
He Jiankui in 2018
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