Amplifying DNA (PCR) Flashcards
lecture 2
What is PCR?
Polymerase Chain Reaction (PCR) is a technique to amplify (copy) DNA by synthesizing new strands complementary to a template strand using DNA polymerase.
Why is PCR important?
PCR is:
Sensitive - amplifies as little as one molecule of DNA.
Specific - amplifies unique sequences depending on temperature and Mg²⁺.
Cheap - cost-effective.
Rapid - results in hours.
Robust - works with old and degraded DNA samples.
What are the three main stages of a PCR cycle?
Denaturation - DNA strands separate (typically at ~95°C).
Annealing - Primers bind to complementary sequences (50–65°C).
Elongation - DNA polymerase synthesizes new DNA strands (72°C).
What components are required in a PCR reaction tube?
Template DNA: The double-stranded DNA to be amplified.
Primers: Single-stranded oligonucleotides (18–24 bases).
Taq Polymerase: Heat-stable enzyme to synthesize DNA.
dNTPs: Building blocks (dATP, dCTP, dGTP, dTTP).
Magnesium (MgCl₂): Cofactor enhancing polymerase activity.
Buffer: Maintains pH and provides salt for optimal reaction.
What are primers, and how are they designed for PCR?
Primers are short DNA sequences that guide DNA synthesis. Good primers have:
Length: 18–24 bases.
G/C content: 40–60%.
Start/end with 1–2 G/C bases.
Melting temperature (Tm): 50–60°C, with primer pairs Tm within 5°C.
No self-complementarity or complementary regions within pairs.
What is Taq Polymerase, and why is it used in PCR?
Taq Polymerase is a heat-stable enzyme derived from Thermus aquaticus. It can synthesize DNA at high temperatures, making it ideal for PCR.
What is the role of magnesium in PCR?
Magnesium (MgCl₂) acts as a cofactor that enhances the enzymatic activity of DNA polymerase, supporting efficient DNA synthesis.
What is the function of the buffer in PCR?
The buffer maintains optimal pH (8–9.5) and provides salts (like KCl or ammonium sulfate) to promote primer annealing and polymerase activity.
How does PCR achieve exponential amplification of DNA?
With each cycle, the number of DNA strands doubles, leading to an exponential increase in the DNA product.
What features of DNA are exploited in PCR?
Hydrogen bonds: Between complementary bases.
Phosphodiester bonds: Form the DNA backbone.
Antiparallel orientation: Ensures correct synthesis direction (5’ to 3’).
How do you detect PCR products?
By running PCR products on an agarose gel stained with an intercalating dye (e.g., ethidium bromide) to determine their size and quantity.
What are the advantages of using Taq Polymerase in PCR?
Heat stability for high-temperature cycles.
High accuracy with only ~1 error per billion bases.
Efficiency in synthesizing DNA at optimal elongation temperatures (~72°C).
What is the optimal temperature for primer annealing in PCR?
Primer annealing typically occurs between 50–65°C, depending on primer design and melting temperature (Tm).
What are dNTPs, and why are they needed in PCR?
Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) are the building blocks that DNA polymerase uses to synthesize new DNA strands.
How does PCR replicate aspects of natural DNA replication?
PCR mimics the process of DNA replication by:
Using primers for initiation.
Synthesizing DNA from a template strand.
Utilizing a DNA polymerase enzyme.
What factors influence PCR specificity?
Primer design and Tm.
Annealing temperature.
Mg²⁺ concentration.
What are the limitations of primers that are too short or too long?
Too short: Lack specificity, may bind to non-target sites.
Too long: Slow hybridization, reduced efficiency.
What is the significance of G/C content in primers?
G/C pairs form stronger bonds due to triple hydrogen bonds, increasing stability and ensuring efficient primer-template binding.
How are PCR products purified for further use?
By using gel extraction or column-based methods to remove unwanted components like primers and buffers.
What is exponential amplification in PCR?
Each DNA molecule is copied in every cycle, leading to a doubling of DNA with each cycle and exponential growth.
