Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

Introduction to Polymerase Chain Reaction (PCR)

PCR Definition

Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify (make multiple copies of) a specific region of DNA. It is a powerful tool in molecular biology and genetics research.

Amplification of DNA

PCR enables the selective amplification of a target DNA sequence, making it easier to study, analyse, or use for various applications.

The Role of Complementary Primers

Complementary Primers

In PCR, short strands of nucleotides called primers are used. These primers are specifically designed to be complementary to the sequences at the two ends of the DNA region to be amplified.

Specific Target Sequences

The primers are critical because they determine which DNA region will be amplified. They bind to the specific target sequences in the DNA.

PCR Process: Repeated Heating and Cooling Cycles

PCR involves repeated cycles of heating and cooling. Each cycle has three main steps, which are carried out at specific temperature ranges:

1. Denaturation (Heating):

The DNA sample is heated to a high temperature, typically between 92°C and 98°C. At this temperature, the hydrogen bonds between the two DNA strands break, causing them to separate into single strands.

2. Annealing (Cooling)

The temperature is then lowered to a range between 50°C and 65°C. During this step, the complementary primers bind to the specific target sequences in the DNA.

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Polymerase Chain Reaction (PCR)

3. Extension (Heating)

The temperature is raised again, usually between 70°C and 80°C. Heat-tolerant DNA polymerase, an enzyme, replicates the region of DNA between the primers, creating a new DNA strand complementary to the target region.

4. Repetition of Cycles

These three steps are repeated for a specific number of cycles, typically 20 to 40 cycles. Each cycle results in a doubling of the DNA in the target region, leading to a significant amplification of the DNA.

Practical Applications of PCR

1. Solving Crimes

PCR is commonly used in forensic science to analyse DNA evidence from crime scenes. It can help identify suspects and provide crucial evidence in criminal investigations.

2. Paternity Suits

PCR can be employed to establish paternity by comparing the DNA of a child to that of a potential father. This is useful in legal cases and family disputes.

3. Diagnosing Genetic Disorders

PCR is used in medical diagnostics to detect specific genetic mutations associated with various genetic disorders. It allows for early diagnosis and treatment.

Example of PCR in DNA Amplification

Let's consider an example where PCR is used to amplify a specific region of DNA for diagnostic purposes:

1. Genetic Disorder Testing

Suppose a patient is suspected to have a genetic disorder due to a known mutation in a specific gene. To confirm the presence of the mutation, a PCR-based test can be conducted.

2. Sample Collection

A sample of the patient's DNA is collected, typically from a blood or saliva sample.

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Polymerase Chain Reaction (PCR)

3. Primer Design

Specific primers are designed to target the DNA region containing the mutation.

4. PCR Reaction

The PCR reaction is set up, including the patient's DNA sample, the designed primers, DNA polymerase, and other necessary components.

5. Cycling

The PCR machine carries out multiple heating and cooling cycles, causing the target DNA region to be amplified exponentially.

6. Analysis

The amplified DNA is then analysed to determine whether the mutation associated with the genetic disorder is present. This information can aid in diagnosis and treatment decisions.

Summary