Methods for Analyzing DNA - Online Tutor, Practice Problems & Exam Prep

Geneticists utilize various techniques to study DNA and RNA, including genomic and cDNA libraries. Genomic libraries contain fragments of genomic DNA, providing insights into non-coding regions and regulatory elements, while cDNA libraries, derived from mRNA, focus on protein-coding sequences. Blotting techniques, such as Southern and Northern blots, separate and visualize DNA and RNA by size using gel electrophoresis. This process involves transferring the separated molecules onto membranes and probing them with specific chemicals to identify sequences, crucial for understanding gene expression and function.

Methods for Analyzing DNA and RNA

So, this is a methods video, so of course, we're going to be talking about different techniques that geneticists use to study DNA and RNA. One type of technique is called a library. Libraries are collections of bacterial clones. This is a bacteria that has been grown and produces the same type of DNA, which is a clone, but it has a ton of different bacteria in it so that we can actually see and extract it, visualize it. They all contain the same DNA and there are multiple of them. Right? So collection, so you have hundreds, thousands of different bacterial clones, and they each contain a different genetic sequence. Now, the type of sequence that it contains depends on how the library was created. You can have genomic sequences, which are going to be small fragments of the genomic DNA, and you also can have cDNA sequences, cDNA libraries, that are made from complementary DNA. Now why would we need both of these? What do these different things tell us? Well, the genomic DNA gives us information about the genomic sequence. Right? And the genomic sequence contains a lot of information that isn't necessarily coding for proteins. It contains regulatory regions, it contains introns, it contains non-coding RNAs. Right? And all these things are found in genomic libraries. When we use the complementary DNA or the cDNA libraries, this complementary DNA is made directly from mRNA. Remember, complementary DNA is reverse transcribed from the mRNA into the complementary DNA. So you start with the RNA of the organism and you turn that into complementary DNA. Well, if you're starting with the RNA, you've already eliminated the introns, the non-coding regions, and the regulatory regions. The complementary DNA libraries represent what the protein-coding region is. What is that sequence? And so, cDNA libraries are super important to look at, what proteins are being expressed at that certain time. And genomic libraries are good at looking at the entire genome, which includes a lot of things that are not protein coding. So that's why we have the two different ones.

How you essentially get a genomic library is you have an organism with some DNA that it could be a cell, it could be a whole bacterium, whatever you want to do. You extract that DNA, and you get it out in a laboratory setting. You digest it with restriction enzymes, so now it's in a bunch of different fragments. And then each of these fragments is put into cloning vectors. This is exactly like the cloning video that is in the other cloning video that we talked about. You put it into vectors, put it into bacteria, and then each one of these is going to be a clone representing each one of these fragments. So there's going to be especially you think of genomic libraries, how big the genome is, that can be hundreds, thousands of bacterial clones that exist. So it's a huge amount of clones representing the DNA found in the genomic library or the cDNA library.

A second method that I want to talk about is blotting. Blotting is super important to separate and visualize and obtain DNA or RNA actually. There are two different types, well, there's more than this, but it's two that we're going to talk about for DNA and RNA. So Southern blots are going to separate the genomic DNA, and then you have Northern blots that are separating RNA sequences. What this essentially looks like is you take a DNA or an RNA sequence, whatever, and you have multiple samples of it. They're likely going to be different sizes. So if we have tube a and it contains like a 5 kilobase sequence, so 5,000 base pairs, and we have tube b and it has 2,000 base pairs and 3,000, even though the total of the sequence is 5, right, because they're different sizes, it's going to separate them differently. You run them on these blots. Essentially, how these blots are run is you take some kind of jelly-like material, almost like jello, essentially. It's called agar, which you may use if you're really into cooking. But, you run these things on these gels, and how they run is they generally run by charge because DNA and RNA are charged molecules. And so if you put a positive at one end and a negative at the other, DNA is negatively charged, it's going to move towards that positive. Now, because it's like jello material, right, that's going to mean it has all of these it's kind of thick, it's hard to get through. So the bigger you are, the less far you're going to be able to travel. So the bigger the DNA sequence, the less far you're going to be able to travel, which is why when you get 5 kilobases, it's up here because it hasn't traveled very far. Whereas, if you get, your 3 kilobytes and your 2 kilobytes pieces, these are going to travel at much lower sizes. And we run these things called ladders that have bands at each one of these, so we know exactly what size it is. And so you run these things on a gel, and then usually for blotting, it's called blotting because you actually have to blot it. And so you have to transfer this onto some kind of membrane, which they again do through charges. But instead of having negative and positive here, if this is your thing, and this is front and back, we do negative and positive this way, where it will run that way onto a membrane. And you don't necessarily, you're not going to be quizzed on these steps, but I am going over them because you may actually do them in a laboratory setting. And so I just want to sort of refresh your memory for this. Then, you transfer this onto a membrane. And then third, the reason we call it blotting is because you blot. And you can blot with different things. There are different chemicals that recognize DNA, some that recognize RNA. There are even some that I'm not

In terms of molecular biology, what is a library?

• A. A place where molecular biology textbooks are stored
• B. A collection of clones containing short genetic sequences
• C. A collection of clones containing a large collection of proteins
• D. A collection of clones with different mutant phenotypes

If you had a sample of RNA to analyze, which of the following techniques would you most likely use?

• A. Southern Blots
• B. SDS-PAGE Blots
• C. Northern Blots
• D. Western Blots

Here’s what students ask on this topic:

What is the difference between genomic and cDNA libraries?

Genomic libraries contain fragments of genomic DNA, which include both coding and non-coding regions such as introns, regulatory elements, and non-coding RNAs. These libraries provide a comprehensive view of the entire genome. In contrast, cDNA libraries are derived from mRNA and represent only the protein-coding sequences. The mRNA is reverse transcribed into complementary DNA (cDNA), excluding introns and non-coding regions. Therefore, cDNA libraries are useful for studying gene expression and identifying which proteins are being produced at a given time.

How are Southern and Northern blots different?

Southern and Northern blots are techniques used to separate and visualize nucleic acids. Southern blots are used for DNA, while Northern blots are used for RNA. Both methods involve running the nucleic acids on a gel electrophoresis, which separates them by size. The separated molecules are then transferred onto a membrane and probed with specific chemicals that bind to the DNA or RNA sequences of interest. The key difference lies in the type of nucleic acid being analyzed: Southern blots for DNA and Northern blots for RNA.

What is the purpose of using restriction enzymes in creating a genomic library?

Restriction enzymes are used to digest the genomic DNA into smaller fragments when creating a genomic library. These enzymes cut the DNA at specific sequences, resulting in a collection of fragments that can be inserted into cloning vectors. Each vector, containing a different DNA fragment, is then introduced into bacteria to create a library of bacterial clones. This allows researchers to study individual fragments of the genome, including non-coding regions and regulatory elements, in a manageable and organized way.

Why is gel electrophoresis important in blotting techniques?

Gel electrophoresis is crucial in blotting techniques because it separates nucleic acids (DNA or RNA) by size. The gel acts as a molecular sieve, allowing smaller fragments to travel further than larger ones when an electric field is applied. This separation is essential for visualizing and analyzing the different sizes of nucleic acid fragments. After electrophoresis, the separated fragments are transferred onto a membrane for further probing, enabling the identification of specific sequences within the sample.

How does a cDNA library help in studying gene expression?

A cDNA library helps in studying gene expression by providing a collection of complementary DNA (cDNA) sequences that are reverse transcribed from mRNA. Since mRNA represents the genes actively being expressed in a cell at a given time, the cDNA library reflects the protein-coding regions of those genes. Researchers can use cDNA libraries to identify which genes are being transcribed and translated into proteins, offering insights into cellular functions, responses to environmental changes, and disease mechanisms.