Lecture 3 - PCR Flashcards
(16 cards)
What is PCR
an in vitro technique that allows the amplification of a DNA
fragment from DNA template
What are the uses of PCR
Research: Detection of specific genes, sequences. Species id.
Medicine: Identification of pathogens (ex: virus!)
Biotechnology: Recombinant systems
Other: Forensics, paternity test, etc
What is PCR based on?
the ability of the DNA polymerase to
synthesize a new strand of DNA complementary to the a
template strand
What is DNA synthesis
sequential addition of deoxyribonucleotides to the 3ʹ-OH end of a polynucleotide chain, the primer strand, that is paired to a second
template strand
PCR stages
Initiation
Denaturation
Annealing
Extension
What is initiation (PCR)
The preparation of the reaction mixture, which includes the DNA template, primers, dNTPs (deoxynucleotide triphosphates), DNA polymerase (usually Taq polymerase), and a buffer solution.
This setup prepares the system for the thermal cycling process
What is Denaturation?
The reaction mixture is heated to a high temperature (usually 94–98°C) to break the hydrogen bonds between the complementary DNA strands, resulting in two single-stranded DNA templates.
Purpose: Separates the DNA double helix into single strands to allow primer binding in the next step.
What is primer annealing?
The reaction is cooled to a lower temperature (typically 50–65°C) to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates.
Purpose: The primers provide starting points for DNA synthesis. The specific temperature depends on the melting temperature (Tm) of the primers.
What is Extension?
- The temperature is raised to the optimal working temperature of the DNA polymerase (usually 72°C for Taq polymerase). The enzyme extends the primers by adding complementary dNTPs to the template DNA strand, synthesizing new DNA.
- This step results in the elongation of the DNA strands, doubling the amount of target DNA with each cycle.
What is amplification for in pcr
- the cycle is repeated 20–40 times, exponentially amplifying the target DNA region
- ensures that there is enough DNA to work with for accurate analysis, identification, and experimentation, especially when the starting material is scarce or degraded
PCR in the lab material and equipment
- Pipettes / tips / ice / gloves
- Eppis and PCR tubes
- PCR instrument
- Electrophoresis
PCR lab - Reaction mix
DNA template
DNA polymerase (thermally tolerant)
DNA polymerase reaction buffer
Mg2+ [MgCl2]
dNTPs [dATP, dCTP, dGTP, dTTP]
H2O
2 primers (forward and reverse)
What DNA polymerases are used in PCR
- Thermus aquaticus -thermophilic bacterium
(Taq polymerase) - Pyrococcus furiosus - Pfu Polymerase -Proof reading activity (3‘→ 5‘
exonuclease) – higher accuracy
Why is MgCl2 used in PCR
- activates DNA polymerase, stabilizes dNTPs, and ensures proper primer-template interaction.
- Proper Mg²⁺ concentration is critical for efficient, specific, and high-fidelity amplification of the target DNA
Too little Mg²⁺: Low amplification efficiency or no product.
Too much Mg²⁺: Non-specific binding of primers and amplification of non-target sequences, leading to low specificity and primer-dimer formation.
What is DNA fingerprinting
It analyzes specific regions of an individual’s DNA that vary highly between people.
These regions are called variable number tandem repeats (VNTRs) or short tandem repeats (STRs) — basically, sequences that repeat a different number of times in different people.
By comparing these regions, scientists can create a unique “pattern” or “fingerprint” of organisms DNA.
How does dna fingerprinting work
Extract DNA from a sample (blood, hair, saliva, etc.).
Use PCR to amplify the STR regions.
Separate the amplified fragments by size using gel electrophoresis.
Compare the pattern of bands to other samples.
Can also use Sanger sequencing instead of electrophoresis to add an extra layer of precision by reading the actual DNA sequence in the repeat regions.
