Polymerase Chain Reaction (PCR) | Zoology Optional Notes for UPSC PDF Download

Polymerase Chain Reaction (PCR): Amplifying DNA

Polymerase chain reaction (PCR) is a groundbreaking molecular biology technique developed by Kary Mullis in the 1980s. It allows the amplification of a single or a few copies of a DNA fragment, generating millions to billions of copies of a specific DNA sequence. The technique relies on the unique properties of DNA polymerase to synthesize a new DNA strand complementary to a provided template strand.

PCR Components

1. DNA Template

   • The sample DNA containing the target sequence to be amplified.
   • Initially, high temperature is used to denature the original double-stranded DNA, separating the strands.
     

2. DNA Polymerase

   • Enzyme responsible for synthesizing new DNA strands complementary to the target sequence.
   • Requires a primer to initiate the addition of nucleotides, as it can only add onto a pre-existing 3'-OH group.

3. Taq DNA Polymerase

   • The most commonly used DNA polymerase in PCR.
   • Derived from the bacterium Thermus aquaticus.
   • Known for its heat resistance, allowing it to withstand the high temperatures of PCR cycles.

4. Primers

   • Short, single-stranded DNA segments that are complementary to the target sequence.
   • Provide the starting point for DNA synthesis by the polymerase.
   • Taq polymerase, used in PCR, extends the DNA from the end of the primer.

5. Deoxynucleotides (dNTPs)

   • Single units of the DNA bases: Adenine (dATP), Guanine (dGTP), Cytosine (dCTP), and Thymine (dTTP).
   • Serve as the building blocks for the newly synthesized DNA strands.
     

6. RT-PCR (Reverse Transcription PCR)

   • A variation of PCR that involves the conversion of sample RNA into complementary DNA (cDNA).
   • Achieved using the enzyme reverse transcriptase.
     

PCR involves a series of cycles, each consisting of denaturation, annealing, and extension steps. During denaturation, high temperatures separate the DNA strands. In the annealing step, primers bind to the target sequences. DNA polymerase then extends the primers by adding complementary nucleotides, creating new DNA strands. This process is repeated through multiple cycles, resulting in the accumulation of the specific DNA sequence in billions of copies (amplicons).

Applications of PCR

• DNA Fingerprinting: Used in forensic investigations to separate and analyze DNA fragments for crime scene investigation and paternity testing.
• Genetic Variation Detection: Identifying genetic variations and proteins associated with health and diseases.
• Nucleic Acid and Protein Detection: Utilized for the detection and purification of nucleic acids and proteins for scientific research.
• Pathogen Detection: Aids in identifying pathogens in blood, tissues, or food sources.
• Proteins and Nucleic Acids Identification: Facilitates the identification and purification of proteins or nucleic acids, often analyzed further using mass spectrometry or DNA sequencing.
• Blotting Methods: Applied in blotting techniques to analyze macromolecules and study evolutionary relationships.
• PCR Result Evaluation: Assists in evaluating Polymerase Chain Reaction (PCR) results.
• Vaccine Development: Plays a crucial role in vaccine development and manufacturing.
• Taxonomy and DNA Profiling: Used in taxonomy-DNA profiling to differentiate species and study evolutionary relationships.

PCR offers a versatile and powerful tool for various applications in molecular biology and genetic research. It has transformed the field by enabling the efficient amplification of specific DNA sequences.

Polymerase Chain Reaction (PCR) Method

PCR is a molecular biology technique that replicates specific DNA sequences. It's based on DNA replication and involves several key components and steps.

Reaction Mixture
The PCR reaction takes place in a laboratory setting and involves combining various components in a sample tube (Eppendorf tube) to facilitate DNA replication. These components include:

• DNA Template: The original DNA to be copied.
• Taq Polymerase: An enzyme, often Taq DNA polymerase, used to synthesize new DNA strands.
• Deoxynucleotides (dNTPs): Building blocks for the new DNA strands.
• Primers: Short DNA segments that bind to the target sequence.
• Other Reagents: Man-made chemical compounds and mix-buffers. The thermal cycler, a specialized machine, is used to control the reaction.

PCR Cycle Steps

1. Sample Preparation (DNA Extraction)

   • Before PCR, DNA must be extracted from a sample, either manually or using specialized instruments.
     

2. Denaturation (Separating the Target DNA)

   • In the denaturation step, the DNA sample is heated to over 95°C, separating the double-stranded DNA into single strands by breaking hydrogen bonds.

3. Annealing (Binding Primers to the DNA Sequence)

   • PCR targets specific DNA sequences marked by primers, which are short synthetic DNA pieces.
   • Two primers, forward and reverse, anneal to each of the separated single DNA strands.
   • Primer binding occurs at temperatures around 50-60°C.

4. Extension (Making a Copy)

   • In the extension step, the temperature is raised to approximately 74°C.
   • DNA polymerase adds nucleotides to the annealed primers, creating a new DNA strand complementary to the template strands.

PCR is a cyclical process where each cycle doubles the amount of target DNA. In around 30 to 40 cycles, more than one billion copies of the target DNA can be generated due to the exponential nature of the reaction.

Reverse Transcription PCR (RT-PCR)

RT-PCR is a variant of PCR used to detect RNA expression. It involves reverse transcription of RNA into complementary DNA (cDNA) using reverse transcriptase, followed by traditional PCR amplification of the cDNA.

Limitations of PCR and RT-PCR

PCR reactions can reach a plateau effect where the exponential amplification stops due to various inhibitors present in the sample. These inhibitors may include reagent limitations, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product. This can limit the amount of target sequence amplified in PCR.