PCR: Principle and applications

Question 1

List four methods of in vitro DNA replication.

Answer 1

(i) polymerase chain reaction
(ii) loop-mediated isothermal amplificaiton
(iii) DNA sequencing reaction
(iii) molecular cloning

Question 2

The most commonly used polymerase in PCR is ____(a)____, named after the heat-tolerant bacterium from which it was isolated, known as ____(b)____.

Answer 2

(a) Taq DNA polymerase
(b) Thermus aquaticus

Question 3

What are the essential items required for PCR?

Answer 3

(a) a template, which is the DNA template (target DNA) containing the target sequence
(b) primers: a forward and a reverse primer to initiate the synthesis of the new DNA strands
(c) thermostable DNA polymerase that carries out the synthesis [the polymerase needs to be heat stable]
(d) deoxynucleotide triphosphates (dNTPs)

Question 4

Outline the steps involved in PCR.

Answer 4

(1) Denaturation
🧬 The double-stranded DNA is heated to around 94 - 98°C for 20 to 30 seconds.
🧬 This high temperature breaks the hydrogen bonds between the DNA strands, resulting in single-stranded DNA.

(2) Annealing
🧬 The temperature is lowered to around 50 - 65°C for 20 to 40 seconds.
🧬 During this step, short DNA primers bind to the complementary sequences on the single-stranded DNA template.

(3) Extension
🧬 The temperature is raised to around 72°C, which is the optimal temperature for the DNA polymerase enzyme.

(4) Repeat cycles
🧬 This denaturation, annealing and extension steps are repeated for 20 - 40 cycles.
🧬 Each cycle doubles the amount of DNA leading to exponential amplification of the target DNA sequence.

(5) Final extension
🧬 After the last cycle, the temperature is maintained at 72°C for 5-10 minutes to ensure that any remaining single-stranded DNA is fully extended.

(6) Hold
🧬 The reaction is then cooled to 4°C to stabilize the amplified DNA until it can be analyzed.

Question 5

List three techniques that can be used in the visualization of the products of a PCR reaction.

Answer 5

◾ Agrose Gel Electrophoresis
◾ Real-Time PCR
◾ Capillary electrophoresis

Question 6

Outline some applications of PCR.

Answer 6

(1) Medical diagnostics
🧬 Infectious diseases: detecting pathogens e.g. viruses, bacteria in clinical samples
🧬 Genetic disorders: identifying mutations and genetic markers associated with inherited diseases

(2) Forensic science
🧬 DNA profiling: analyzing DNA samples from crime scenes for identification and comparison
🧬 Paternity testing: establishing biological relationships through DNA analysis

(3) Agriculture
🧬 Genetically Modified Organisms: Detecting and characterizing GMOs in crops and food products.

(4) Genetic research
🧬 Phylogenetic studies: Analyzing genetic relationships and evolutionary history among species.