DNA-Based Diagnostics PDF

PROPRIETARY. DO NOT SHARE. Transcript: How Diagnostic Tests Work: DNA-Based Diagnostics Section 1: Polymerase Chain Reaction (PCR) Technology Welcome Welcome to our course on How Diagnostic Tests Work: DNA Based Diagnostics. This course will be broken into four sections: Polymerase Chain Reaction (...

PROPRIETARY. DO NOT SHARE. Transcript: How Diagnostic Tests Work: DNA-Based Diagnostics Section 1: Polymerase Chain Reaction (PCR) Technology Welcome Welcome to our course on How Diagnostic Tests Work: DNA Based Diagnostics. This course will be broken into four sections: Polymerase Chain Reaction (PCR) Technology, Microarray Technology, Next Generation Sequencing (NGS) Technology, and microRNA Technology. Section 1: Polymerase Chain Reaction (PCR) Technology Objectives At the end of this section, you should be able to: • Describe the use of DNA probes in diagnostics. • Describe PCR (Polymerase Chain Reaction). • Explain the use of PCR in diagnostic applications. DNA Structure Let's start by reviewing the structure of DNA. DNA, as you recall, stands for deoxyribonucleic acid. DNA is made up of core units called nucleotides. A nucleotide has a phosphate group, a sugar (deoxyribose), and a base, which will either be thymine, guanine, cytosine, or adenosine. A nucleotide is composed of all of these. DNA Sequence That's what's shown here at the bottom of the Main Topic. It's the schematic of a nucleotide where the base is thymine. In DNA, the nucleotides are connected one to the next by phosphodiester bonds which is a chemical bond, a connection, between the phosphate group and the two sugars. So, DNA is just a string of nucleotides, one connected to the next. What changes are the bases? So here we have the bases as G T G A C T. This order of the bases is referred to as the DNA sequence. The information in the DNA is contained in that sequence. A gene is a specific sequence of DNA. If I change the sequence, I change the information. Complementary Base Pairing What you see here is a single strand of DNA, but in the cell, DNA occurs as a double strand. The double strand is created by what's known as complementary base pairing. It turns out in nature that G always binds to C and T always binds with A. These are what we call base pairs. DNA 1 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. occurs in your cells as a double strand and the double strand is composed of bases that form complementary base pairs: A to T, G to C. Again, the order of the bases whether it's A-C-T or CA-T, is where the information is found in the DNA. That order in which the bases, that is the nucleotides, are connected one to the next is known as the DNA sequence. It's the DNA sequence that can give us diagnostic information. Diagnostic DNA When we talk about diagnostic DNA then, what we want to do is to locate and identify specific sequences of DNA. In order to do that, we use what's called a probe. A probe is a synthetically or chemically produced short sequence of DNA. Probes are also known as oligonucleotides. Oligo just means a few, so we have a few nucleotides. Probes can be anywhere from six to usually not more than fifty nucleotides long. Mostly they're in the ten to thirty nucleotide range. A probe is a very specific sequence of DNA that is made chemically in the lab. DNA Probes This sequence of DNA, this probe, is complimentary to a target sequence of DNA that appears in the cell. They match up to each other. The probe, in addition to being chemically synthesized and composed of nucleotides, also generally has a label on it, something that allows us to identify it, that allows us to visualize it. This could be with a fluorescent dye, something that when we shine a light on, it fluoresces. The probe will complimentary base pair A to T, G to C. The probe will complementary base pair with our target sequence of DNA, which is the sequence of DNA that we want to use for diagnostic purposes. We want to detect the presence or absence of that particular sequence of DNA. Once the probe binds, we can then detect our label and see whether it is in the cell. If the probe doesn't bind, it gets washed away. There's no signal. That particular target sequence is not present. Probes can be used to detect specific DNA sequences in tissue samples, in quantitative PCR, or on a DNA chip. We're going to go over each of these different categories in the next sets of slides. Sequence-Specific Viral Detection If, for example, we wanted to detect the presence or absence of a particular virus, we're going to look at the DNA sequence of the virus. One important thing to note is to use the probes, we have to know the sequence of that virus or bacteria, or gene that causes a disease ahead of time. 2 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. There has to be a database that contains the sequence that we're trying to detect. That organism has to have had its DNA sequenced. We'll discuss how we sequence DNA later in the course. Sequence-Specific Viral Detection - Sensitivity Sequence-specific detection is the most sensitive means of diagnosing a viral infection or of detecting contamination in different products. This could be a food product, or this could even be a therapeutic product like a vaccine, or an antibody used to treat a disease. The supposition here is that each organism has its unique DNA sequence. We are simply going to detect that sequence. If we detect the sequence, if we find it to be present by a probe binding to it, then we know that a particular organism or virus is present. If the probe doesn't bind to it then we know we don't have that contamination or that disease. The use of DNA sequencing is so sensitive that it allows us to detect a virus in a patient's blood before virus-specific antibodies are present. This is because, while the virus may be there, there may not be a high enough viral load to trigger an immune response. Your immune system has not made antibodies against it yet. However, using our DNA diagnostic we can detect minute amounts of DNA of that virus. Even though your immune system hasn't responded, it could be there. In using a DNA diagnostic, we will be able to detect its presence. Methods for DNA Sequence Detection The two most common techniques for the detection of specific sequences of DNA are polymerase chain reaction, or PCR, and a variation of PCR called QRT-PCR. These are fairly simple to do and have been well-established since the 1990s. They require some equipment to run, and PCR is relatively inexpensive, while qPCR and qRT-PCR are a little more expensive but gives us more information. Polymerase Chain Reaction (PCR) First, let’s take a look at all the components required for PCR. Click each button to learn more about these components. 3 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Thermocycler We start with what's called a thermocycler. We place our DNA sample in a tube and then place that tube in the thermocycler. This is a machine that heats up and cools down the DNA. It heats our DNA to 95-100 degrees centigrade. This temperature is high enough to cause our DNA to separate into two single strands. The DNA we are examining could be the patient's DNA or could be viral or bacterial DNA isolated from the patient. In either case, the DNA sample is the DNA in which we are searching for our target sequence. Tube Reagents We then add it to the tube reagents. Our first reagent is our DNA primer. That's also what we would call our probe. These again are sometimes called oligonucleotides. These are short sequences of DNA that we have specified what the sequence is. They are produced chemically. That sequence determines where we start copying the DNA and when we stop copying the DNA. We also add in a taq polymerase, that's the DNA copying enzyme. And lastly, we add in free nucleotides, that are unattached A's, G's, T's, and C's because that's what the taq polymerase uses to fill in the template. The next screen is an animation that illustrates our PCR reaction. DNA amplification will only occur if the target DNA – the particular gene sequence we are looking for, or the specific viral or bacterial DNA – is present. DNA Tools - PCR A PCR reaction is set up to contain a small amount of sequence DNA: two oligonucleotide primers, nucleotides, and the heat-resistant DNA Polymerase (taq polymerase). The mixture is placed in a thermocycler, a machine that controls the temperature of the PCR reaction. The DNA is heated to 94 degrees Celsius causing the double-stranded DNA to separate. The reaction is then cooled to 42 degrees Celsius, allowing the primers to hybridize into the complementary sequences in the DNA. The DNA present between the two primers is the only portion of the DNA that will be amplified. This section of DNA is called the target. The reaction is heated to 72 degrees Celsius, the optimal temperature for taq polymerase to amplify DNA. This completes cycle one. Cycle 2 is the same process repeated. Double-stranded DNA is separated into two single strands: the primers hybridize and taq polymerase amplifies the DNA. During each subsequent cycle, the number of DNA copies grows exponentially. Until over thirty cycles, over a billion copies of the target DNA are made. 4 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Quantitative PCR A variation of PCR is something called qPCR or quantitative PCR. This allows us to calculate how much of a particular gene or piece of RNA is present. Frequently qPCR is used to detect how much RNA is present, not only if the RNA is there. qPCR An example of the use of qPCR is trying to detect bacterial meningitis by looking at a gene unique to bacterial meningitis, a DNA sequence called ctrA. In this case, we are using taq DNA polymerase. This is the heat-stable DNA polymerase enzyme that will not denature even though we're heating it and cooling it down. It's able to copy the DNA. Then we have what's called EXPRESS SYBER GreenER. This is a fluorescent dye that is incorporated into the DNA as we run the PCR reaction so that as we get more and more pieces of DNA, more and more dye is incorporated, and we can detect that. What happens is that as we copy more and more DNA, we can measure a larger and larger fluorescent signal because SYBER Green is a fluorescent dye. If we have our particular piece of DNA or our piece of RNA present, the signal increases. Notice that line going up is straight? Anytime we have a straight line we can make all kinds of calculations based on the slope and the intercept. That allows us to determine concentrations as well. qrRT-PCR qRT-PCR is the PCR that is used specifically to detect how much RNA is present. The Q stands for quantitative, and the RT stands for reverse transcriptase. What reverse transcriptase does is copy RNA back into DNA. Reverse transcriptase is an enzyme that was found only in viruses and scientists and companies were able to purify the reverse transcriptase for use in these PCR systems. Now you can buy it separately and use reverse transcriptase to copy any piece of RNA that you want to. Why is it that we want to copy RNA into DNA? It's because RNA is chemically unstable. If we don't copy it quickly it will degrade, and we will lose some or all of our sample. DNA on the other hand is quite stable. Once we copy our RNA into DNA, we now have a stable template with which we can work. The reverse transcriptase enzyme does the copying by reading a single strand of RNA and copying it into a piece of DNA. This DNA is called cDNA, which stands for copied DNA. 5 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. For example, if we want to detect the flu virus, the flu virus has a particular protein called HA protein, the Hemagglutinin Protein. Any protein is coded by a gene. Well, it turns out in the flu is an RNA virus. Its genes are composed of RNA instead of DNA. Therefore, we take a set of primers or probes that define the beginning and end of the HA gene in our flu virus. We then copy that RNA sequence using our reverse transcriptase into cDNA. We now have a cDNA copy of the flu RNA gene. Then we begin to copy that into other copies of DNA by doing our PCR using taq polymerase in the presence of our EXPRESS SYBER Green. We've copied the flu RNA into flu DNA. The more flu RNA we have, the more cDNA we are going to have. As we begin to amplify that cDNA using our taq polymerase in the presence of SYBER Green, we're going to get a green, fluorescent signal. Once again, if we get a positive signal, if the fluorescence increases, we know that the flu virus is present. Section 1: Polymerase Chain Reaction (PCR) Technology Summary To summarize, in this section we learned that: • A DNA probe is a synthetically or chemically produced short sequence of DNA that is complementary to a target DNA sequence, allowing scientists to detect the presence or absence of a gene of interest. • PCR is used to amplify or make a large number of copies of a very specific region of a genome of a DNA sequence allowing for visualization of that DNA sequence. PCR is very specific and works quickly. • Using fluorescent dyes PCR can be quantitative allowing for the diagnosis of diseases that could be caused by over or under-expression of a particular gene. 6 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Microarray Technology Section 2: Microarray Technology Objectives At the end of this section, you should be able to: • Define SNIP (Single Nucleotide Polymorphism). • Describe the use of SNPs in microarray technology. • Discuss the use of SNP chips in diagnostic applications. Single Nucleotide Polymorphism (SNP) First, we need to define SNPs. SNPs are single nucleotide polymorphisms. This is a one-base difference between different genes. This is where we change an A to a T, or an A to a G, or an A to a C. In our example below, we see that we have a DNA sequence A-C-T-C-G-C- T-T-C-G. We see that in that whole sequence, we've changed a T to a C. That's a single base change or a single nucleotide polymorphism. One base is changed. In our introduction to diagnostics, we discussed the consequences of this, which is simply that a one-base change could change the amino acid sequence of the protein, which could then change the structure and function of that protein. That may cause a disease, or it could change how fast or how slow a particular enzyme works, leading to the need to change how a drug is dosed. We would like to be able to detect what particular version of a gene we have, and what SNP we have. As with this example, do we have the T version or the C version? SNP Chip To determine what SNP is present, one method is to use an SNP Chip. We could also do this using our PCR reactions, but PCR reaction doesn't allow us to have coverage of lots of different SNP possibilities. Instead, we use an SNP Chip. This is sometimes called a microarray. Here we have a picture of a microarray. This is a gene chip from Affymetrix, also sometimes known as hybridization assays. We're basing this on the principle that we have a probe or a singlestranded piece of DNA that contains the sequence we're interested in detecting. In this case, our single-stranded DNA is physically attached to a surface, usually a specially treated glass plate. DNA Assays – aka Hybridization Assays This glass plate is subdivided into multiple grids. It's like a giant Excel spreadsheet. That's what's 7 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. represented here. In each of those little squares, we can put a different sequence of DNA corresponding to a different SNP, a different single nucleotide polymorphism. By knowing the location of each square, just like in an Excel spreadsheet, cell A-1 or cell Z-53, whatever it is, we know which DNA sequence we have attached to the surface in that particular part of the grid. When anything binds to that, we can detect it, and we can say we see a signal in cell B-35. We've attached to that the CY2D6 SNP. We know that that is the particular version of that gene that we have based on where we get hybridization or complementary base pairing. Let's look at an example of that. Here we've taken one particular cell out of our grid, and we've attached our single-stranded DNA sequence to this cell. Notice they are all the same. We want lots of copies of the sequence, so we get enough hybridization, enough complementary base pairing, that we're able to get a signal that we can detect. We have a specific DNA sequence attached here. We take the side-by-side chip and attach a slightly different sequence in order to detect sequence differences. We then take a patient's DNA and attach a fluorescent label to that DNA and mix it into our hybridization chip. There's a little injection port on that Affymetrix chip that we showed you. The sequence then migrates through the chip until it finds its complementary base pair. Binding G’s to C's, T’s to A's, and that particular sequence binds. SNP Detection When we shine a fluorescent light, or a laser light over the whole chip, wherever binding has occurred, and wherever the two strands have complimentary base-paired, we get a fluorescent signal. We can see that on the chip because it glows. Then there is a CCD camera that takes a picture of this, and that is compared to the original grid. Based on the location of where we get a signal or where we get a color response, we know what sequence was bound. That tells us which particular version of an SNP we have. SNP Chip Example Another example of the use of an SNP chip is looking at the ApoE (pronounced one word A – POE) gene. This is the e4 SNP. This is a gene that gives us a prediction of whether or not you are predisposed to Alzheimer's disease. You can see here, we've accounted for each possible variant, which is for each possible SNP. T, A, C, and G. We're going to take the patient's DNA, add it to our SNP chip and if it binds in our first grid, the one closest to us, we get our signal there. If we have a different sample with a different SNP that binds in another grid, based on the location 8 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. of that binding, we can determine whether you have a particular risk. So, if you have both A's then there's no e4 allele or gene, which means you are at the least risk for getting Alzheimer's. If you get an A-G sequence match, then you have one of the alleles. Your risk of getting Alzheimer's increases slightly. If you have a G-G, then you have both of the e4 alleles. You have a fairly high risk of getting Alzheimer's disease. So, this is a diagnostic based on your DNA. We're looking at your sequence. We're seeing which version of the SNP you have. Based on that version we can predict what your risk is, in this case, of developing Alzheimer's disease. Of course, it works for other genes that we have similar types of data about. Remember, once again, this is a data issue. We have to have sequenced the gene from normal patients and patients with the disease. We're going to compare the sequences of normal patients to those who have the disease and look for differences in the nucleotides in their DNA sequence. Based on that, if we always see the same particular base occurring in one position in the diseased person, then that becomes a diagnostic for the disease. We can create a SNP chip or a PCR reaction or another method to detect that particular DNA sequence. SNP Chip Output The output for an SNP chip is just a series of colors. This is captured by a CCD camera. By looking at what grids light up we can see which sequences are complementary to the patient's sequences. That tells us which variant of the SNP the patient has. The computer generates the colors for us, prints out the data, and then we can compare this to the database. The computer will also start to make some predictions and so forth. The advantage of these chips is that we can check or interrogate hundreds of thousands of sequences all at once. Some of these SNP chips will hold 500,000 different DNA sequences, which means I can check or interrogate 500,000 different SNPs at one time. The SNP process takes usually about half a day to a full day to complete. However, what's replacing our microarray chips, which have some reproducibility issues, is something called next generation sequencing. First generation sequencing was developed in the mid to late 1970s. It was very slow, very tedious. It could take up to two years to obtain accurate sequence information for just 500 bases of DNA. In contrast, next generation sequencing is fairly automated sequencing. It uses something called massively parallel sequencing where we simultaneously sequence lots of pieces of DNA. We can sequence 1 to 10 million bases per day. Next generation 9 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. sequencing or NGS will be discussed in the next section of the course. Section 2: Microarray Technology Summary To summarize, in this section we learned that: • SNPs are single nucleotide polymorphisms, which means one base difference between different genes • SNP chips can detect hundreds of thousands of DNA sequences simultaneously, which allows for data on the sequence of many genes at once, something that can be particularly useful when trying to diagnose any genetic differences between normal physiology and a disease state. 10 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Next Generation Sequencing (NGS) Technology Section 3: Next Generation Sequencing (NGS) Objective At the end of this section, you should be able to: • Explain the purpose of whole genome sequencing. • Discuss the applications of whole genome sequencing in diagnostics. • Describe the advances being made in next generation genome sequencing. Next Generation Sequencing (NGS) A currently hot market is next generation sequencing. Several companies are in the running for this. One is Illumina, which makes the HiSeq (PRONOUNCED HIGHSEEK). There are different versions of the HiSeq depending on what your budget is. Then we have ThermoFisher, which does ion proton, and also the solid system. What varies between these instruments is, one, how much DNA they can read per sample. With Illumina and ThermoFisher, the HiSeq versus the ion proton is 150 base pairs to 200 base pairs. Again, the base is just the base of A's, G's, C's, and T's. You can look at the instrument cost. The HiSeq was significantly higher than either of the ThermoFisher products. All of these take one day to do a genome. The SOLiD takes seven days because you notice it does only 75 base pairs. But the accuracy of the SOLiD system is greater than either the ion proton or the HiSeq. We expect that these costs will continue to drop over the years. Originally, to sequence one human being's genome was over one billion dollars. Now we're down to $1000 and we can do this in a day. NGS: Reversible Dye Terminator Let's look at how next generation sequencing works. One variant that we talk about here is the reversible dye terminator. All of the DNA sequencing system methodologies rely on the ability to copy or duplicate DNA. Essentially what we're doing when we are sequencing is something very similar to DNA replication or very similar to PCR. We're going to take a single-stranded piece of DNA. We're going to use that as our template. Then by complementary base pairing, we're going to determine the order of the nucleotides with reversible dye termination. What we're going to do is we're going to start copying the DNA with our DNA arrays. It is a requirement before you can copy DNA that you have to have a little starter piece of double-stranded DNA. This would be our 11 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. probe. This probe defines the sequence that we want to start to copy. We put the probe in our sample, get our double-stranded piece of DNA, then add in free nucleotides, each of which has a different fluorescent dye on it, a different colored fluorescent dye. You can see that we have A's, G's, C's, and T's. In addition, our free nucleotide not only has a fluorescent dye, but that fluorescent dye is in a position that prevents any additional nucleotides from being added on. We can't connect the next nucleotide while the dye is there. We start our sequencing. If you look here, the first based on our single-stranded DNA is a T. What will be complimentary to that is an A. We're going to incorporate an A into our short probe chain there that makes it double-stranded. Now we have A matched with T. Any further incorporation, or any further copying stops because we have that termination chemical on our A. We do a little reaction and wash away all the other nucleotides. We then break off the little termination chemical. Now that A is free. Now it behaves as if it is a normal nucleotide. We can add it to the next base. As we remove the dye, we also in essence bleach out that nucleotide. Now it becomes colorless. We get a reading. Once we've read it, we know that based on its color, it's an A. We then add a chemical that removes the terminator, which is the chemical that blocks the incorporation of the next nucleotide and bleaches out the color. We add in our mixture of all four nucleotides because remember, as we're sequencing, we don't know what the next base is. Here is an illustration we do, so you can watch what happens. In our illustration, you see the next base is a C. What will be complementary to a C is a G. Now G has been incorporated. It connects to the A. We get the appropriate color for the C. We do our readout. We flash the light on there and get the fact that it's a G. Then we add in our chemical to remove the terminator and bleach out the G. Now we're ready to incorporate the next nucleotide, which we see on our single chain is an A. The complementary nucleotide is, what? a T. T comes in. It gets incorporated. We rinse away all of the other nucleotides again after we read the color, bleach out and remove the terminator. Now we're ready to incorporate the next and so on. We just keep doing this over and over again until we have sequenced all of the DNA. We can do this in a single tube. This works out quite nicely, and it's a high throughput method. We'll just let the reaction go to the end here. All we do is we do this on multiple pieces of DNA. Then using some fairly sophisticated software we then assemble all the fragments into one continuous piece. We've cut up this DNA into fragments where the pieces will overlap. Using the appropriate algorithm that is the appropriate mathematical equations, the computer will determine 12 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. for us where the overlapping sequences are and assemble them in the correct order to give us a continuous genome. NGS: Ion Torrent The other type of next gen sequencing we want to talk about is called ion torrent. This is a pretty clever method. The other type of next gen sequencing we want to talk about is called ion torrent. This is a pretty clever method. Again, we're copying a piece of DNA using DNA arrays. Here what we're going to take advantage of when we copy our DNA sequence is the fact that when we incorporate a nucleotide, it releases a proton, a little hydrogen ion that is positively charged. This is detected. There's a specially constructed matrix that we call an ion-sensitive layer that creates an output, an output little electrical current that's detected by the ion sensor whenever we get an incorporation even. Every time a nucleotide is added in, we get a little signal. What happens here is that the next nucleotide is a T, so we would expect that an A would be incorporated. We start by adding T into the tube. T won't incorporate, so we rinse away the T. We add an A. A will incorporate so now an ion is released. We rinse away an A. The next nucleotide we have is a C. We start by adding T first all by itself. No reaction occurs. We rinse away the T. We now try an A. Again, no reaction occurs because A is not complementary to C. We rinse away the A. We add in a C. C is not complimentary to C, so we rinse that away. Then we go to our fourth choice, a G. G is complimentary to C. It gets incorporated and that results in the release of an ion. We detect a signal. The next one is an A. What will be incorporated? a T. So, we get a signal. Another T will be incorporated, and we get three signals at once. We know that we have three Ts incorporated. Then we have a G. The next one would be a C and so forth. This is a little bit cheaper because we're just measuring the release of an ion. That is much less expensive than having to work with fluorescent dyes, which can become fairly expensive. NGS: The Future of Medicine The concept here then, in next generation sequencing is that both of these companies, because of the increased speed of sequencing, are hoping that hospitals and other types of facilities that 13 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. use diagnostics will incorporate sequencing as a routine part of the diagnostic process. We're looking at things like sequencing the DNA from tumor cells and sequencing people's DNA to determine an appropriate dosage of a drug or appropriate therapeutic use. At the same time, we're going to add to the database of DNA sequences of both healthy and diseased individuals, which will help us to identify the important DNA sequences that may be relevant to the disease. IlluminaDx, in which Dx is our symbol for diagnostics, has a cancer discovery initiative. Here our biomarker discovery validation and diagnostic product development will be DNA sequences. One of the advantages of this is that with PCR and next generation sequencing, we can obtain these results very quickly, relatively cheaply, and with a high level of sensitivity. By sensitivity, we mean we can detect very small amounts before most of the other methods will detect anything. It means the sooner we can detect the presence of a disease, the sooner we can begin to initiate an effective treatment for the disease. Additionally, these methods will help prognostics, which is determining the course of the disease. Section 3: Next Generation Sequencing (NGS) Summary To summarize, in this section we learned that: • The hope is that in the future genome sequencing might be used as a routine part of the diagnostic process. Sequencing things like DNA from tumor cells, or genes that code for enzymes that break down drugs to determine an appropriate dosage of a drug or appropriate therapeutic use. • 14 Whole genome sequencing could also create a database of DNA sequences of both Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. healthy and diseased individuals, which will help us to identify the important DNA sequences that may be relevant to the disease. • Reversible termination and ion torrent sequencing are two next generation sequencing technologies that could lead to much faster and more accurate whole genome sequencing. 15 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 4: microRNA Technology Section 4: microRNA Technology Objectives At the end of this section, you should be able to: • Define microRNA. • Discuss the potential applications of microRNA in molecular Diagnostics development. microRNA Diagnostics The last technology we want to talk about is the amazing field of microRNA. MicroRNAs are short pieces of RNA that do not code for proteins. MicroRNAs, it turns out, are regulatory RNAs. These are RNAs that control the expression of genes. When we say control the expression of a gene, we mean they control whether the gene is going to be activated or not, that is whether the gene will make its protein product. Whether we go from DNA to RNA to protein or not. There are thousands of microRNAs. They are involved in the development of stem cells, and they are also implicated in a number of cancers. One of the ways that microRNAs work is they are complementary to a particular target RNA which is a messenger RNA that contains the information that's going to make a protein. A singlestranded messenger RNA is simply a copy of a gene containing the information to make a protein. That messenger RNA goes to the ribosome. The ribosome reads the information on the messenger RNA and then makes the appropriate protein. If we can prevent that messenger RNA from getting to the ribosome, then the information won't be read, and the protein won't be made. That's one of the places where microRNA plays a role. When the microRNA binds to the target messenger RNA, we have double-stranded RNA. The cell detects the double-stranded RNA. It sees it as a threat because some viruses have doublestranded RNA as their genetic material. The cell thinks it's being invaded by a virus, and it degrades that RNA. One of the things scientists are working on quite a bit is understanding the profiles of the microRNAs. We have to one, determine the sequence of all the microRNAs. There's still a lot of work to be done with that. It is important to determine how much microRNA is made because in part the amount of microRNA determines the extent to which a gene will be expressed. Then we also need to identify which genes each specific sequence of microRNA regulates. These pieces 16 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. of microRNA tend to be fairly short. They are basically about the same length as a probe, usually about 20 nucleotides in length. microRNA Diagnostics - Advantages Differences in the amount of microRNA have been linked to a difference between normal cells and cells that have become cancerous. This may be used as an early detection method for certain types of cancers. It turns out that microRNA, even microRNAs related to internal cancers like pancreatic cancer, liver cancer, or lung cancer, have been detected in the saliva of patients. We are looking at the concept of analyzing people's saliva for microRNAs in order to have a diagnosis of cancer. This will be a very sensitive method to detect very small amounts of cancer-associated microRNAs before any other diagnostic may pick them up, especially traditional imaging diagnostics. MicroRNA for these tumors may appear when it's at a very small, minimal cell stage, which could make it very easy to treat. Section 4: microRNA Technology Summary To summarize, in this section we learned that: • MicroRNAs are short pieces of RNAs that do not code for proteins but are regulatory RNAs. • Detecting variations in microRNA levels shows promise as potential cancer diagnostic and may also be useful as a detection method for pathogens, for disease-causing organisms. • The advantage of using microRNA to detect disease could be that these tests could be extremely sensitive because of our ability to detect very small amounts of microRNA. This could lead to earlier diagnosis over traditional methods and could also allow for non-invasive testing with only a sample of the patient’s saliva needed. 17 Copyright 2023 Biotech Primer, Inc.