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DNA Amplification – Analytical Techniques in Biotechnology

by Kevin Ahern, PhD
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    00:01 It is said that knowledge is power and with biotechnology we can see in this presentation how it is true where the knowledge about molecular biological processes has been brought to develop powerful analytical techniques.

    00:13 I am going to talk about three of those techniques in this presentation.

    00:16 DNA amplification, microarray analysis and 2D gel analysis.

    00:21 Now, knowledge about the process of DNA amplification has been applied in a technique called the polymerized chain reaction that has revolutionized the way that we see and analyze DNA.

    00:33 The polymerized chain reaction allows a person to amplify specific DNA segments out of a larger DNA millions of times.

    00:42 It's very popular for a forensic analysis and if you ever watched a crime show on TV you have probably seen PCR being applied to action.

    00:51 The process of PCR or polymerized chain reaction is derived simply by copying some ideas from cellular DNA replication and then using this in a noble way to accomplish the replication of DNA that one desires only to copy in a specific way.

    01:09 The advantage of PCR is that it is a very simple technique to perform A high school student can be trained to do PCR in about an hour.

    01:17 It requires sequence knowledge to start and that's one of the primary requirements of PCR, as we shall see.

    01:25 Now the PCR technique uses the knowledge of the sequence to design and chemically synthesize DNA primers flanking the region to be amplified. Now I need to explain that.

    01:36 In a previous lecture I talked about how DNA polymerase uses a primer to start DNA replication and in the cell that primer is RNA.

    01:46 Now the problem with the RNA primer is, it’s got to be removed and then replaced by something else. That's kind of a complicated process.

    01:53 If we wanna replicate DNA in a test tube and we wanna do it efficiently, We start with a DNA primer.

    01:59 And the beauty of a DNA primer is that it defines a starting point for replication.

    02:04 As since a primer has a specific sequence one can design that sequence and then allow it to find the right place to form base pairs and that will define where the replication is occurring, as we shall see.

    02:17 To bring this process about, we have to take a target DNA. So this DNA might be let's say a human DNA and this human DNA contains a region that we are interested in analyzing.

    02:28 Maybe it's a genetic mutation that a person has suffered that we are interested in determining what it is.

    02:33 In each case we would know the sequences around the DNA that we are interested in studying, so, let's says, for example, we have a person who has sickle cell anemia.

    02:43 And sickle cell anemia involves a mutation within hemoglobin gene.

    02:48 We know the sequences of the hemoglobin gene but we don't know the sequences of the mutation in the middle of the gene.

    02:54 So we could design DNA primers that are complementary to either end of the hemoglobin gene, make those and make them so that they will now form base pairs with the end of the hemoglobin gene within the DNA.

    03:08 We take that target DNA which is the person who has the mutation we take their DNA, mix it with the primers, mix it with a 4 dNTPs and here is the kicker, a thermostable DNA polymerase. Now we will see in a minute why that thermostable DNA polymerase is important. The thermostable part means that it's resistant to denaturation by heat. Most enzymes fall apart in heat, a thermostable one doesn't.

    03:39 We take this whole system. This mixture that I have just defined to you and we add it to something called a thermocycling system. And what a thermocycling system is, it is a device that will heat up this sample and then cool it to various temperatures as we shall see.

    03:54 And so this begins a process that I am going to describe next.

    03:58 So we can see for example here the target DNA of the person that has the mutated hemoglobin gene on top.

    04:04 Its a duplex DNA and I have made it short here.

    04:09 But the reality is that we would have the entirety of that person's chromosomes. So there is a lot of sequences here.

    04:15 The first step in the process of employing PCR is to pull the strands apart.

    04:21 Now this kind of mimics what the helicase was doing in DNA replication expect for here we are physically pulling them apart.

    04:28 But how to do pull them apart? The way that you pull them part that you heat the mixture to near boiling.

    04:33 When you do that, the hydrogen bonds that hold the DNA duplex together are broken and the strands completely come apart. So the strands separate here.

    04:43 The second part is to cool that temperature down so that the primers that were mixed in the solution can form base pairs with their appropriate sequence.

    04:53 Now if you make the primers of the right length, the only place is where they will form the base pairs are where you intended them to, on either side in this case of the hemoglobin gene.

    05:04 This process requires a specific temperature called an annealing temperature.

    05:09 So the thermocycler heated the system up to boiling and now it's cooled down to this magical temperature where the primers will form base pairs with their complements.

    05:22 The third step then is for the DNA polymerase to replicate those strands.

    05:27 Well, the DNA polymerase was already in the mixture and remember we used a thermostable DNA polymerase and because of that it didn't get damaged by that boiling that we did.

    05:39 The DNA polymerase uses the 4 dNTPs to replicate the strand of interest and we can see the replication now that is occurring here.

    05:47 So after that's happened the primers defined the ends. So I see one end in yellow and I see another end in green.

    05:58 This replication proceeds and now what happens is the primers direct replication over and over and over of the same strands.

    06:07 So notice that the two strands that we're copying are just the top and the bottom strand. They are actually the same sequence.

    06:13 So then we get a duplex that's made from that.

    06:16 It means that we started with one duplex in step 1.

    06:20 But by step 3 we have two copies of that same duplex.

    06:25 But you can start to see what happens here. Every time we do a cycle of boiling, annealing and replicating, we double the number of strands.

    06:35 Now that might not seem like a big deal. But if you do 30 times you have 2 to the 30th more strands than you started with at least in theory. That's over a billion.

    06:48 So that means you can take very tiny amounts of DNA and make incredible quantities out of it using this method. This is why it is a very powerful tool in a crime scene.

    06:56 It doesn't take very much of a suspect's DNA to get this kind of analysis done.

    07:02 This process is typically repeated for 30 to 40 cycles although there are some processes where it's done even more.

    07:10 Now this powerful technique is one of many that we use to analyze sequences. So for example analyzing DNA is done by PCR but there are other types of analyses that we are interested in doing. And these types of analyses I'd like to sort of lump together and call Omics analysis.

    07:28 We will see how that comes into play in just a moment.

    07:30 The technological advances really are what have made the Omics analysis possible.

    07:36 Omics methodologies focus on individual molecules within a cell but across a broad spectrum.

    07:44 So what happens for example that if we talk about genomics, there is one of the Omics, we are studying every DNA sequence in a cell.

    07:52 Now 30 years ago that was inconceivable, nobody knew how to do that now we can sequence a genome in very short periods of time, a week.

    08:02 We can do transcriptomics in which we are analyzing all of the transcripts of a cell.

    08:07 Now, the transcripts of course are the RNAs that are being transcribed for making protein.

    08:13 If we know all of the RNAs of a cell and how many of each is being made we have an amazingly broad piece of information about what the cell is doing and how much of it it's trying to make.

    08:26 Proteomics is another one of the Omic that's involved in the study of protein expression.

    08:33 So just like we can use transcriptomics to tell us how many and what kinds of RNAs are being made, proteomics allows us to determine how many and how much of each protein is being made in a cell.

    08:47 So metabolomomics is an analysis of the metabolome, a metabolome of course corresponds to all of the metabolites that are being made in a cell.

    08:55 That could include all of the molecules made in a citric acid cycle and what quantity of each is being made. All of the molecules being made in glycolysis and what the quantities of each of those are.

    09:05 In other words, all of the different biochemistry that is happening spectrally across the entire cell. Now that's the advantage of Omics type analysis and there are dozens of Omics disciplines now that people have developed to do these kinds of analyses.

    09:21 And as a result of this we are actually able to understand at a system level what's happening in the cell. Instead of understanding at an individual molecular level what's happening in the cell and what happens with that system analysis allows us to better understand what life is really all about.


    About the Lecture

    The lecture DNA Amplification – Analytical Techniques in Biotechnology by Kevin Ahern, PhD is from the course Analytical Techniques in Biotechnology.


    Included Quiz Questions

    1. It has three steps to each cycle
    2. It requires an RNA primer for each round of replication
    3. It works best with a heat-sensitive DNA polymerase
    4. It typically amplifies DNA about 1000 fold
    1. denaturation is done first
    2. Renaturation must be done very quickly
    3. Elongation must be done at temperatures near boiling
    4. Actual doubling of the amount of DNA occurs with each cycle
    1. RNA primer
    2. DNA template with target sequence
    3. Four types of dNTPs
    4. Taq polymerase
    5. DNA primers
    1. Denaturation of template DNA — done with the help of thermostable helicase enzyme in the PCR mixture
    2. DNA primer — determines the replication start point during PCR
    3. Taq polymerase — a thermostable polymerase enzyme resistant to the denaturation step during PCR
    4. Thermal cycler — a device used to amplify the target DNA sequence via PCR
    5. Annealing temperature — temperature which affects the efficiency and specificity of PCR system by facilitating the hybridization of primer to the DNA template
    1. The detection of genes, mRNA, proteins, and metabolites in a specific biological sample
    2. The replication of target genes in a very short time
    3. The synthesis of target mRNA with high accuracy and efficiency
    4. The synthesis of target tRNA and rRNA with high accuracy and efficiency
    5. The synthesis of the target protein and its related metabolites with high accuracy and efficiency

    Author of lecture DNA Amplification – Analytical Techniques in Biotechnology

     Kevin Ahern, PhD

    Kevin Ahern, PhD


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