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Background:

From Genomes:

The temperature of a PCR mixture is first set at 94 °C so that denaturation of DNA can happen. Then it is reduced to 50–60 °C, which results in some rejoining of the single strands of the target DNA, but also allows the primers to attach to their annealing positions. After this DNA synthesis begins as the temperature is raised to 72 °C, the optimum for Taq polymerase.

In this first stage of the PCR, a set of ‘long products’ is synthesized from each strand of the target DNA. These polynucleotides have identical 5′ ends but random 3′ ends, the latter representing positions where DNA synthesis terminates by chance. [Subsequent cycles give] rise to ‘short products’ whose 5′ and 3′ ends are both set by the primer annealing positions.

Now Wikipedia says:

[...] Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.

Questions:

  1. I’m wondering that if the shortening of the length of new DNA is purely by chance. From Wikipedia it appears that the length of the DNA synthesised can be controlled by the temperature of the mixture?
  2. To carry out a PCR experiment, the target DNA is mixed with Taq DNA polymerase, a pair of oligonucleotide primers, and [...]

A pair of primer cannot support the increasing number of PCR cycles, are primers added later/ throughout the process?

  1. Besides it seems to me that the temperature of the mixture has to be changed according to the phases of the cycle. Is it changed? How is it determined that which phase of the cycle the DNAs are in?

P.S. I'm not sure if this post should be broken down into two posts or more. Suggest if you feel that's necessary.

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  1. Ideally amplification happens between the two primer pairs and results in one product. However, complicated secondary structures, the depletion of some of the reagents may cause pauses and fall off of the polymerase. This happens often later in the reaction and results in shorter products. This final amplification step is introduced to finish all these shorter products.

  2. A pair means the combination of a forward and a backward primer (see the image below from here). Bothg primers are added in a concentration high enough that both are not depleted and enable long PCR runs. No primers (or other reagents) are added during a standard PCR reaction.

enter image description here

  1. The temperature of the PCR reaction is changed through 1 reaction cycle: First you start with the denaturation of the DNA at 94°C, then cool down to anneal the primers (typically between 50 and 60°C, depending on the primers), followed by the amplification step at 72°C (it'S length depends on the length of the amplified DNA piece, Taq polymerase processes about 1kb/min) (see figure below from here). This is one cycle and you know where you are, because the PCR machine counts this cycles.

enter image description here

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