During which step in the PCR cycle are nucleotides used?

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During which step in the PCR cycle are nucleotides used?

PCR cycle

PCR is an enzymatic reaction that relies on DNA polymerase in the presence of template DNA, primers, and four deoxyribonucleotides.

PCR increases the number of DNA fragments exponentially by subjecting the DNA fragment to be amplified and its complementary oligonucleotide strand primers on both sides to multiple cycles of a three-step reaction of high-temperature denaturation, low-temperature annealing, and primer extension.

This results in the desired large number of specific gene fragments in a short period of time.

Competition PCR is a kind of quantitative PCR. The concentration of the target template is determined by adding an artificially constructed competition template with mutations to the PCR reaction system and controlling the concentration of the competition template to make a quantitative study of the target template.

The PCR technique is used in combination with the restriction enzyme digestion technique. The PCR amplification product of the target gene is cleaved with the restriction enzyme, and the polymorphism of the gene is detected by analysis of the cleaved product.

High specificity The determinants of specificity of PCR reactions are.

• the specific and correct binding of the primer to the template DNA.
• the principle of base pairing.
• the fidelity of the Taq DNA polymerase synthesis reaction.
• the specificity and conservativeness of the target gene.

Initial denaturation

Complete denaturation of the template DNA and complete activation of the PCR enzyme is critical to the success of the PCR. Refer to the reagent instructions for recommended heating times, generally two minutes for unmodified Taqase activation.

Denaturation steps in the cycle　　

Normally 95°C for 30 seconds is sufficient to completely denature various target DNA sequences in the cycle, but this step can be shortened if possible.

Too long a denaturation time damages the enzyme activity, while too short a denaturation of the target sequences is not complete and may result in amplification failure.

Primer annealing

The annealing temperature should be determined from various aspects, generally based on the Tm value of the primer as a reference, and adjusted downward according to the length of the amplification as the annealing temperature. Then make a prediction based on this experiment.
　　
The annealing temperature has a great influence on the specificity of PCR.

Primer extension　　

Primer extension is generally performed at 72°C (the optimal temperature for Taqase). However, this step can be omitted when the amplification length is short and the annealing temperature is high.
　　
The extension time depends on the length of the amplified fragment and is generally recommended to be above 1000 bp, with a setting of 1 min/kbp for derivatives containing Pfu and its derivatives.

Nucleotides

Nucleotides are phosphate esters synthesized by the dehydration condensation of pentose hydroxyl groups in a nucleoside or deoxyriboside molecules with inorganic phosphoric acid. Those produced from nucleosides are called nucleotides; those produced from deoxyribonucleosides are called deoxyribonucleotides.

Since there are three (2, 3, 5), free hydroxyl groups, on the pentose ring of nucleoside, three types of nucleotides can be produced: 2-adenosine, 3-adenosine, and 5-adenosine; there are two (3, 5) free hydroxyl groups on the pentose ring of deoxyribonucleoside, only two types of nucleotides can be produced, such as 3-deoxyadenosine and 5-deoxyadenosine.

The nucleotides described above all contain a phosphate group and are collectively referred to as nucleoside monophosphate (NMP). However, the phosphate group of 5-nucleotide can be further phosphorylated to produce the corresponding nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP).

The structures of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) are shown below.

Correct Answer

Here is the correct answer to During which step in the PCR cycle are nucleotides used?

During the extension step in the PCR, cycle are nucleotides used.

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