PCR - Basics, Types, and Math!
Polymerase chain reaction (PCR) is a method used to amplify genetic material. This article will cover the basics of PCR, types of PCR, and how to solve PCR math problems.
The Basics
PCR has 3 steps: denaturation, annealing, and extension. Denaturation is done at a high temperature to separate the strands of DNA. Annealing allows primers to stick to the ends of each strand. Extension is done by a heat-compatible polymerase like Taq.
Types of PCR
There are three main types of PCR to know for the USABO: normal PCR, quantitative PCR (qPCR), and reverse transcriptase PCR (RT-PCR).
qPCR is also called real-time PCR. Through fluorescent markings on every piece of DNA generated, we are able to quantify the rate of PCR over time. The most straightforward method uses SYBR green, a fluorescent marker that binds to double-stranded DNA. Thus, each time DNA replicates, so does the fluorescent intensity. We can also use the TaqMan probe method. These probes bind to a segment of the single-stranded DNA prior to the extension step. When the polymerase reaches where the probe is, the probe is cleaved by the polymerase’s exonuclease activity. This activates the fluorescence in this probe, and we’ll get a measurable signal that again can be plotted over time. One application of qPCR is that the time it takes for one DNA segment to reach a measurable threshold versus that of another segment gives you information about their initial abundances.
RT-PCR begins by converting RNA to cDNA using reverse transcriptase. Like other polymerases, RT requires primers. In this case, we can use a poly-dT primer complementary to the poly-A-tail of the RNA. We can also insert random hexamers (hexa=6, so 6-nucleotide segments) to bind throughout the RNA (RT has low processivity, so we need a lot of primers to prevent it from falling off). Once the cDNA is created, it can remain single-stranded, but it’s also possible for a second strand to be created, and the PCR happens from the ds cDNA instead. But ss cDNA is more common.
Math of PCR
Here’s what PCR looks like with a focus on a certain target DNA sequence:
Notice how some strands are just the target sequence, some have one long end, and some have a complete strand. This is due to the strict 5’-3’ directionality of the polymerase. Some questions will ask, after n cycles of PCR, how many of each group are there? Here are the formulas for these.
First: there are always 2 complete-length strands, no matter the value of n.
Second: there are 2n one-sided strands.
Third: there are 2^(n+1)-2n-2 only-target strands. This is because after n cycles, there will be 2^(n+1) total strands. Then we subtract the former 2 groups from that.
So yeah here’s some videos:
General PCR https://youtu.be/JmveVAYKylk
RT-PCR https://youtu.be/1vqNZ-H7Pq0
Helps understand the math concept https://youtu.be/JmveVAYKylk?si=a7ECZHSlsKq65NZ8
