During which step in the PCR cycle do primers form bonds with a single-stranded template?

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During which step in the PCR cycle do primers form bonds with a single-stranded template?

PCR

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be thought of as a special DNA replication outside of a living organism, and the most important feature of PCR is its ability to increase trace amounts of DNA dramatically.

Thus, whether it is fossilized paleontology, the remains of a historical figure or the hair, skin, or blood left behind by a murderer decades ago, as long as a tiny bit of DNA can be isolated, PCR can be used to amplify it and compare it. This is where the power of “trace evidence” comes in.

First conceived by Mullis in 1983, the invention of the polymerase chain reaction, or simple DNA amplification, in 1985 meant the real birth of PCR technology.

PCR takes advantage of the fact that DNA denatures to single-stranded at 95° Celsius in vitro. At low temperature, the primer binds to the single strand according to the principle of base complementary pairing and then adjusts the temperature to the optimal reaction temperature of DNA polymerase, which synthesizes the complementary strand along the direction of phosphate to five-carbon sugar (5′-3′).

The PCR instrument based on polymerase manufacturing is actually a temperature control device that can control well between denaturation, denaturation, and extension temperatures.

Principles of PCR

The semi-conserved replication of DNA is an important pathway for biological evolution and transmission. Double-stranded DNA can be denatured and unspooled into single strands by the action of various enzymes and replicated into the same two molecular copies according to the principle of base complementary pairing with the participation of DNA polymerase.

In experiments, it was found that DNA can also undergo denaturation unstranding at high temperatures and can be repurposed into double-stranded when the temperature is lowered.

Therefore, by controlling the denaturation and denaturation of DNA through temperature change, DNA polymerase and dNTP can be added to design primers to complete the in vitro replication of specific genes.

However, DNA polymerase is inactivated at high temperatures. Therefore, new DNA polymerase has to be added in each cycle, which is not only tedious but also expensive, limiting the application and development of PCR technology.

The discovery of the heat-resistant DNA polymerase-Taq enzyme was a milestone for the application of PCR. The enzyme can withstand high temperatures above 90°C without inactivation and does not require enzyme addition per cycle, making the PCR technique very concise. It also greatly reduced the cost, and PCR technology was applied in large numbers and gradually used in clinical applications.

The basic principle of PCR technology is similar to the natural replication process of DNA. Its specificity depends on oligonucleotide primers that are complementary to both ends of the target sequence.

The PCR cycle consists of three basic reaction steps: denaturation-annealing-extension.

1) Denaturation of template DNA.

The template DNA is heated to about 93°C for a certain time to dissociate the double-stranded template DNA or the double-stranded DNA formed by PCR amplification to make it single-stranded so that it can bind to the primers in preparation for the next round of reaction.

2) Annealing of template DNA and primers.

After the template DNA is denatured into single-stranded by heating, the temperature is lowered to about 55°C and the primers are paired and bound to the complementary sequences of the single-stranded template DNA.

3) Extension of primers.

The DNA template-primer conjugate is synthesized under the action of 72°C and DNA polymerase, such as Taq DNA polymerase, with dNTP as the reaction material and the target sequence as the template, and a new semi-conserved replicated strand complementary to the template DNA strand is synthesized according to the principle of base complementary pairing and semi-conserved replication. Repeat the denaturation-annealing-extension process to obtain more “semi-conserved replication strands”. This new strand can then be used as a template for the next cycle. It takes 2-4 minutes to complete each cycle, and 2-3 hours to amplify the target gene several million times.

Correct Answer

Here is the correct answer to During which step in the PCR cycle do primers form bonds with a single-stranded template?

During the Annealing step in the PCR cycle, primers form bonds with a single-stranded template.

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