In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges

In a typical PCR reaction, describe what is happening in...

Key Concepts

Denaturation

During the denaturation phase, typically at temperatures around 92-95°C, the double-stranded DNA molecules are heated to break the hydrogen bonds between the complementary bases, resulting in the separation of the DNA into two single strands. This step is essential for making the strands accessible for primer binding in subsequent steps.

Annealing

In the annealing phase, temperatures ranging from about 45-65°C allow short pieces of single-stranded DNA, known as primers, to bind to their complementary sequences on the single-stranded DNA templates. This binding is crucial as it defines the starting point for the new DNA strand synthesis.

Extension

The extension phase occurs at temperatures around 65-75°C, during which the DNA polymerase enzyme adds nucleotides to the 3' end of the annealed primers, synthesizing new strands of DNA. This step results in the formation of a complete double-stranded DNA molecule, completing one cycle of amplification.

Transcript

00:01 This question asks, suppose you are working in a molecular biology laboratory and are having difficulty performing the pcr successfully.

00:09 You decide to double check the pcr protocol programmed into the thermocirer and discover that the annealing temperature was programmed to be 65 degrees centigrade instead of 50 degrees centigrade, as you had intended.

00:22 What effects would this mistake have on the pcr reaction? to answer this question, let's first focus on what the polymerase chain reaction.

00:31 Reaction or pcr reaction entails.

00:34 So there are going to be three main steps during the polymerase chain reaction.

00:39 We have the first step, which is denaturation, that occurs at roughly 95 degrees centigrade.

00:53 Then we have the annealing phase, which occurs at about, in this case, we wanted to occur at 50 degrees centigrade.

01:05 And then the elongation phase that occurs at 72 degrees centigrade.

01:16 So the annealing phase in our problem occurs at 65 degrees centigrade instead of the 50 that was intended.

01:27 And due to this, we are seeing some problems.

01:31 So we know that during denaturation, we have double strand separation.

01:39 During the annealing phase, we have primer annealing or primer addition, where the primer is going to stick to our single strand.

01:57 And then elongation is where we have our nucleotide addition.

02:03 Then after the elongation phase, we loop background and perform the denaturation phase, and this cycle continues on for about 25 to 40 cycles...

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