Lab 8 Quiz - PCR

Polymerase Chain reaction

rapidly produces millions to billions of copies of a specific segment of DNA

PCR applications

• sequencing

• gene cloning and manipulation

• gene mutagenesis

PCR Principles (6)

1. DNA of interset

2. DNA Polymerase

3. Two DNA primers

4. DNA nucleotide bases

5. buffer solution

6. bivalent cations - Mg or Mn

Thermal cycles

series of 20-40 repeated temperature changes

Individual steps (4)

1. initialisation

2. denaturation

3. annealing

4. extension/elongation

Step 1. Denaturation

DNA template heated to 94-96ºC breaking weak hydrogen bonds

• creates a single stranded DNA

Step 2. Annealing

cold to 50-70ºC to let the primer bind to complementary sequence

Step 3. Extension/Elongation

heated to 60-72ºC so DNA polymerase can extend the primer and add nucleotides

PCR advantages (5)

1. high sensitivity

2. can test for anti-microbial resistance

3. quick

4. cost-effective

5. ability to detect less common organisms

PCR disadvantages (5)

1. lower specificity

2. requires narrow list of causative agents

3. may amplify normal flora

4. less cost-effective when using multi-organism approach

PCR method used in lab

Real Time PCR (qPCR)

qPCR

accurate and sensitive with a fast turn around time

qPCR is used to diagnose (4)

1. UTIs

2. wound infections

3. nail fungal infections

4. STIs

PCR vs qPCR

• qPCR is quantitative with a very high resolution

  • quantifies particular fragment in sample

• PCR is qualitative with very poor resolution

  • detects presence or absence of certain genomic fragments