My Fascination with Science - PCR Tests - 5. Cycle 2 - All three steps

 

So, this is the result from the end of the first step. We then begin the cycle of denaturing, extension and annealing.

When denatured, they separate. For convenience, I have reorganized the strands. I have also added the primers to the right end for brevity.

Here is how things will look when extended. I have added a number to each of the strands for clarity in the next step. Look at strands 5 and 7. This is exactly the secuence that we are interested in. When annealed, at the end of the second step, we will have one double stranded DNA fragment of interest, just like that!

This is the beauty of the whole process. But wait until the next step.

My Fascination with Science - PCR Tests - 5. Cycle 1 - Step 3 - Extension Step 1

Extension - This is where the exciting thing happens where copies of the snippet are made. I suggest you be patient and go through each of these steps carefully yo understand whats going on. You will find some stuff repeated from before for completeness.

Here is the separated strands of a denatured DNA with the 5' and 3' ends marked and the snippet we want to copy is indicated.

Here is where I show the primers paired with the single strands of the denatured DNA. This association happens during the annealing step.

In the extension step, polymerase goes to work. In short, DNA polymerase looks for 3' end of a single stranded fragment of a DNA and adds complimentary nucleotide to make double stranded DNA. This is important to understand. For one, you need to have a single stranded potion of the DNA and secondly you need a 3' end open for the extension. As you see in the diagram below, this is exactly what the primer addition provides for both of the separated strands of the original DNA. Secondly, you need a tons of nucleotide for polymerase to add. So, in this step, the scientists add a ton of nucleotide. The temperature where this takes place is also very important it is typically in the mid 70's. DNA Polymerase is a generic term and there are a lot of different polymerases. The one that fits the bill here becaue of the temperature is a naturally occuring polymerase called Taq Polymerase. So, the original DNA, primers, LOTS of nucelotides, and Taq polymerase constitute the mixture. For reasons of stability and efficiency etc. additional chemicals such as a buffer are added. Scientists have figured out that this step should be carried out in the 72 - 76°C.

When done, you will get this.

That completes the first cycle! At the end of this, you have two double stranded DNA with some portions remaining single stranded in one of the two strands. This whole step of mixing everything, denaturing, annealing and extending all takes a few minutes.

In the next post we will look at the subsequent steps. 

My Fascination with Science - PCR Tests - 4. Cycle 1 - Step 2 - Annealing

Annealing - This is a step where you are going to attach two "stops", one each, to the individual strands with the idea that these primers will form double helices (very small ones indeed) at the start and end of the snippets.

Remember that you should know which specific snippet you want to copy. Based on that you create two small single stranded sequences called Primers. We want them to pair up with the beginning of the snippet on one strand and the end of the snippet on the other strand, like shown below.

Credit: Addgene

Remember that pairing them up means creating the primers to have the nucleotides with the complementary bases chained together. For example, say the four bases at the end of the snippet you want to copy is AGGC in the 3' to 5' direction, then the primer that will pair with it should be TCCG, but in the 5' to 3' direction and so on.  This is just an example. Primers are longer generally. Your primers are constructed synthetically by stitching together a sequence of bases that are complimentary to the ends of the snippet you want to copy. As a result they are specific to the snippet you want to extract.

Choosing a proper primer is a complicated business. You can read about how to design best primers here. You want the primers to connect just to the snippet you are interested in and not elsewhere. Given that there are millions of bases in a DNA, there is a high likelihood that unless carefully chosen, there may be other places it will latch on to, something we really don't want. The temperature at which you anneal depends on the primer you choose. It is roughly 3 times the # of G-C pairs plus 2 times the # of A-T pairs you will have when primers bind to the DNA. By carefully reducing the temparature based on the nature of the primer, you get the primers to attach to the single strands in the right place. 

Primers may also attach elsewhere because of the nature of things, but they may not attach exactly the same way, but a little weaker, for example. This is why the annealing temperature is important and chosen in such a way that such weaker connections will be blown away.

----------------------------------------------------------------------------------------------------

Details that are relevant but you may choose to skip...

Because of the complimentarity of the strands, the sequence of nucleotides youu see in the top primer will be identical to the sequence in the bottom strand of the original DNA. In other words if you were to drop vertical lines at the ends of the top primer down, the base sequence in the bottom strand in the same location will be identical. This is a very important thing to understand from the perspective of how replication/copying will work to produce the snippet.

Here is a bit more colored version of the situation:

• Here are the denatured DNA strands. I have used a random sequence of A, T, G and C for illustrative purposes. I indicate the 5' and 3' ends for each strands. If you look, each base from the top strand will have its complementary base at the bottom. I show some 50 basees in each strand and as I have said before, these are millions of base long, so the dots indicate that.

• In this figure I indicate the snippet that we wish to copy. Again, totally arbitrary.
  
  
• In the figure below, I show examples of two primers. Again, primers are longer than the 6 I used here and they don't necessarily have to be identical in length. However, they must have annealing temperatures that are very close or identical.
• The primer for the top strand is selected to pair with the end of the snippet.  The 6 bases of the snippet is TGCGAC in the 5' to 3' direction. The primer is ACGCTG, but in the 3' to 5' direction. Now, you go vertically down, you will see the same exact 6 base arrangement in the original DNA strand.
• Similarly, the primer that pairs with the strand at the bottom is attaching itself with the "beginning of the snippet". The original strand has TCTTTG as the 6 bases, but because it is the complimentary strand, the direction is 3' to 5'. The primer for it is AGAAAC in the 5' to 3' direction. Look up vertically and you will see it to be identical to the original DNA strand.
• This is obvious once you see it with examples, but not necessarily easily understood with schematic diagram. This is the beauty of complemntarity and the whole PCR procedure takes advantage of it.

My Fascination with Science - PCR Tests - 3. Cycle 1 - Step 1 - Denaturation

Denaturation - Bascially, separate the two stands of DNA. Through various trials, scientists have found that around 94 -96°C the two strands separate while retaining their structural characteristics. Think of the double helix as a zipper and this step bassically unzips the DNA. It is important to understand that each strand of the DNA has a particular direction and for all the rest of the steps, the direction plays a very important role. I have added more details regarding the directionality to the bottom of this for those who are interested.

The zippers we are used to have ends, but in some sense they are identical. But, imagine zippers where the top and bottom stops can be one of two choices, A and B. The left side of the zipper has A and B respectively for as its top and bottom stoppers and the right side has B and A instead. It is likely to be useless as a zipper, but this is the analogy with DNA. Of course, to complicate things further, each tooth of the left zipper can be one of four choices and magically, the right zipper where the top and bottom are reversed has a tooth at every step that is complementary to the left one and they nicely come together. 

Credit: lumen learning

----------------------------- Details that you can skip if not interested

Directionality:

As I wrote earlier, each strand has a particular direction if you look at the atomic level. At some point, the DNA that is millions of base pairs long needs to have ends. The structural characteristics of the end is used to denote diretion. Here is the depiction of a nucleotide and millions of these are connected together to form the DNA. 

Credit: National Human Genome Institute

As you can see, a nucleotode consists of a Phosphate group, a sugar molecule, and one of the bases we discussed earlier (A, T, G or C).

Now, if you look at the detailed figure, you will see a numbering system for the carbon atoms in sugar. The end of a helix where the Phosphate group looks this way (it has this OH group that is the "end") is connected to the carbon designated as 5', that is referred to as the 5' end of a strand. Similarly the O atom you see that is connected to the Carbon designated as 3' turns to OH, that will be called the 3' end.

Credit: Wikipedia

My Fascination with Science - PCR Tests - 2. Some Basics

PCR Stands for Polymerase Chain Reaction. Think of it as a clever copy machine that can create amplified copies of a certain portion of a very dense page and it can produce millions and millions of them in a short time. Where PCR has revolutionized science is in making millions or billions of copies of portions of DNA (genetic material in organisms) quickly. Whenever you think about chemistry and "quickly" it will involve an enzyme, which accelerates chemical reactions. DNA Polymerases are enzymes that help synthesize DNA fast from their components.

Credit: Wikimedia

From Wikipedia - "DNA is is a molecule composed of two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses" . In very simple terms, DNAs are extremely long, consists of two strands and have millions of nucleotides.

Each of the two strands is wound as a helix and both are connected using what are referred to as "hydrogen bonds". As complex as they are, they are composed of just four bases (part of a nucleotide), called Adenine (A), Thymine (T), Guanine (G) and Cytosine (C). Further, the base in one strand of the helix dictates what is in the other - A & T are always paired and G and C are always paired. Each strand has a unique directionality to it that are opposite of each other. This is critical in the PCR process. I will talk about this in the next few posts.

Despite the fact that we have only four nucleotides and two unique pairs (four if you want to be strict), the number of combinations you can create when there are millions of them is huge and this is what gives rise to complexities and intersting variations in DNA. 

There are numerous applications where we need to concentrate on a small snippet of this humongously long chain. For example, this small snippet may be relevant to detecting particular diseases, or is unique to a criminal that needs to be matched against samples from a crime scene, or unique to a particular bacteria or virus which can be used to find if someone is infected with a specific disease. Scientists have identified such unique snippets in DNAs of various organisms by now thanks to rapid advances in technology. However, samples tend to have very small quantities of DNA and copying them to get millions of identical copies helps simplify further analysis. 

We have a DNA sample with millions of bases, but depending on the application, we are only intereted in a small snippet of it. Wouldn't it be cool if we can simply make copies of just that portion in enough quantities that it can be easily analyzed further? This is exactly what PCR does. It does this in three steps that are then repeated multiple times:

• As a zeroth step, understand clearly the nucleotide sequence of the snippet that you want to copy.
• Denaturation - where you basically separate the two strands of DNA by heating.
• Annealing - where you add synthesized fragments called Primers. One primer forms the second (complementary) helix at the beginning of the snippet of interest with one of the denatured strand and another which pairs up with the end of the snippet on the other. You make them attach this way by carefully cooling the system.
• Extension - This is where the polymerase swings into action by extending one end of the primer with nucelotides that has been added. At the end of one cycle, you will now have two double helical DNA, but with overhangs of single strand DNA, because of where the primers are and how the polymerase only extends in one direction.
• You repeat this process whereby during each cycle, you double the amount of DNA. But, the beauty of this process, as you will see later, is that after the second cycle, one of the DNA that will get copied this way will be just the snippet of interest. It turns out that after about 30 cycles you will likely have a billion copies, Yes A BILLION! And guess what, this whole process has been automated and each cycle only takes a few minutes each and the entire process can be completed in a few hours. 

Kary Mullis invented this and if you are interested, you can read about him here and I encourage you to watch this video. He invented this in 1983 and received Nobel Prize in Chemistry for this in 1993. I should caution you that there are many videos of him on YouTube, but be careful in choosing which one to watch. At least a couple I watched, while funny, got into how he doesn't believe that there is a global warming issue and I don't want tht to cloud his excellent contributions :)

My Fascination with Science - PCR Tests - 1. Introduction

I missed out on learning a lot of things when I was a grad student, though opportunities existed, for two primary reasons.

• It was extremely hard to be living away in a country that is so different from where I had lived before and away from my family and I was unable to concentrate on studies.
• When I could concentrate, I wanted to devote all my time to learn everything that was there to learn about the work in the research group I wanted to work. I knew exactly who I wanted to work with, so unlike other grad students, I did not do any rotation, wait for the completion of course work etc.

Though my research at the tail end of my graduate studies involved computer simulation of small fragments of DNA and then as a postdoc larger systems of DNA and proteins, my contribution was more. on the compuational aspects, data management and analysis and visualization. Whereas I had basic understanding of structures and chemistry, it was not as deep as I would have liked it to be.

But my fascination about molecular biology never left me and though I was not reading published scientific work in peer reviewed journals, I was still reading about new discoveries or watching documentaries etc with keen interest. 

Then, I decided to take the course called Introduction to Biology - The Secret of Life on edX during the Fall of 2013. It was taught by a group led by Eric Lander, Founding Director of the Broad Institute; Professor of Biology at MIT. He is currently a Presidential Science Advisor and Director of the Office of Science and Technology Policy for the Biden administration. This was a fantastic experience and I thoroughly enjoyed.

I was not engaged and in fact I hated a similar course that I had to take as a grad students, primarily because of bad teaching. But Eric is a terrific teacher and the entire course was so engaging and I was so excited. I would even tell my wife not to accept any social engagements on weekends when quizzes were due or exams were scheduled. I passed with flying colors :)

I took courses in several other areas such as Quantum Computing, but over time, I could not sustain the time and effort it needed. Roll forward to 2019... COVID struck and we started hearing about PCR tests and though by this time I had some understanding of it, if someone asked me to explain in detail and how it is useful in detecting COVID viruses, I would have struggled.

So, I invested time to try to understand this at a level of depth that I needed to. What follows in the following posts is my personal understanding of how this works bassed on several videos I have watched and several explanations I have read on the web. I will spare you a comprehensive list. Here are a couple that I found to be at the right level of detail:

• Understanding PCR - Mark Temple 
• PCR (Polymerase Chain Reaction) - Amoeba Sisters
• Polymerase chain reaction (PCR)

If you are an expert in this area and feel that you can add a comment to help simplify this and help readers who are not as well versed in the science behind this, please do.