Question

Describes the steps of how a PCR generates many copies of a particular sequence.

Answer 1

There are three main stages:

• Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
• Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
• Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

A complete PCR reaction can be performed in a few hours or even less than an hour with certain high-speed machines.

After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

Denaturing stage

• During this stage, the cocktail containing the template DNA and all the other core ingredients is heated to 94-95?C.
• The high temperature causes the hydrogen bonds? between the bases in two strands of template DNA to break and the two strands to separate.
• This results in two single strands of DNA, which will act as templates for the production of the new strands of DNA.
• It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely.
• This usually takes 15-30 seconds.

Annealing stage

• During this stage, the reaction is cooled to 50-65?C. This enables the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding (the exact temperature depends on the melting temperature of the primers you are using).
• Are primers single strands of DNA or RNA? the sequence that are around 20 to 30 bases in length.
• Are the primers designed to be complementary? in sequence too short sections of DNA on each end of the sequence to be copied.
• Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases.
• The two separated strands of DNA are complementary and run in opposite directions (from one end - the 5’ end – to the other - the 3’ end); as a result, there are two primers – a forward primer and a reverse primer. This step usually takes about 10-30 seconds.

Extending stage

• During this final step, the heat is increased to 72?C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.
• Taq DNA polymerase is an enzyme taken from the heat-loving bacteria? Thermus aquaticus.
• This bacteria normally lives in hot springs so can tolerate temperatures above 80?C.
• The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
• DNA polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37?C (body temperature).
• 72?C is the optimum temperature for the Taq polymerase to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.
• The result is a brand new strand of DNA and a double-stranded molecule of DNA.
• The duration of this step depends on the length of DNA sequence being amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb).

These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA sequence of interest.

The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase enzyme can attach and start making DNA.

A result is a huge number of copies of the specific DNA segment produced in a relatively short period of time.