Question

Explain how PCR (polymerase chain reaction) works. After explaining PCR, compare PCR to the DNA replication that occurs naturally in living cells, making sure to give at least 4 similarities and 4 differences.

Answer 1

Answer- Polymerase chain reaction (PCR) is a method widely used in molecular biology to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation.

The individual steps common to most PCR methods are as follows:

Initialization:

This step is only required for DNA polymerases that require heat activation by hot-start PCR. It consists of heating the reaction chamber to a temperature of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases are used, which is then held for 1–10 minutes.

Denaturation:

This step is the first regular cycling event and consists of heating the reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.

Annealing:

In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.
It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature.

Elongation:

The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq (Thermus aquaticus) polymerase is approximately 75–80 °C (167–176 °F),[13][14] though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are complementary to the template in the 5'-to-3' direction

Similarities between normal and DNA replication and PCR-

• Both these processes lead to synthesis of new DNA molecules.
• Both processes needs DNA polymerase enzyme.
• Both of these needs deoxyribonucleic acids.
• Both processes operates at optimum temperature range.

Differences between normal DNA replication and PCR-

• Normal DNA replication process is a cellular process which occurs in our body and is crucial for cell division while PCR technique is used for amplification of DNA and its occurs in lab conditions (artificial environment).
• The rate of DNA replication in normal body is very less as compared to that of PCR technique.
• In PCR technique we can use different kind of polymerase according to our need but DNA replication in normal body is carried out by specific DNA polymerase only.
• By using PCR technique we can specifically amplify the specific segment of DNA but a normal DNA replication of our body the complete DNA is replicated.