PCR: Principle and applications Flashcards
List four methods of in vitro DNA replication.
(i) polymerase chain reaction
(ii) loop-mediated isothermal amplificaiton
(iii) DNA sequencing reaction
(iii) molecular cloning
The most commonly used polymerase in PCR is ____(a)____, named after the heat-tolerant bacterium from which it was isolated, known as ____(b)____.
(a) Taq DNA polymerase
(b) Thermus aquaticus
What are the essential items required for PCR?
(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)
Outline the steps involved in PCR.
(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.
List three techniques that can be used in the visualization of the products of a PCR reaction.
◾ Agrose Gel Electrophoresis
◾ Real-Time PCR
◾ Capillary electrophoresis
Outline some applications of PCR.
(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.
Which of these statements is incorrect with regard to Polymerase Chain Reaction (PCR)?
(a) It requires two primers.
(b) It requires a single primer.
(c) It allows the DNA from a selected region of the genome to be amplified several times.
(d) A cycle of PCR has 3 steps: Denaturation, annealing and extension.
(e) It can use a DNA or RNA template.
(b) It requires a single primer.
Which of the following is not a required component in a PCR?
(a) Template DNA
(b) Pair of Primers
(c) dNTPs
(d) Thermostable DNA Polymerase
(e) DMSO
(e) DMSO
Further notes:
Dimethyl sulfoxide is not a required component in a PCR. It is sometimes used as an additive to improve the amplification of certain templates, but it is not essential for the reaction to occur.
What would be the effect on the PCR reaction if any of the following circumstances arose?
1. Lack of primers in the reaction
2. Lack of dNTPs in the reaction
3. No Taq polymerase in the reaction
(a) PCR would proceed normally.
(b) Non-specific PCR of random templates will occur.
(c) The reaction will cease after a few cycles.
(d) The PCR reaction will not commence.
(e) The primers would dimerize and be extended to generate a single albeit small fragment.
(d) The PCR reaction will not commence.
Which of the following statements are true regarding PCR?
(a) Billions of copies of the desired DNA segment can be made from nanogram quantities of template DNA.
(b) Automated PCR machines are called thermal cycles.
(c) A thermostable DNA polymerase is required.
(d) Mg2+ are required as co-factors for the enzyme.
(e) All of the above.
(e) All of the above.
The process of binding the primer to the denatured strand of DNA during PCR is called ________.
(a) denaturation
(b) renaturation
(c) annealing
(d) strand extension
(e) none of the above
(c) annealing
Which of the following is true about PCR?
(a) Denaturation involves heating to 90°C - 98°C.
(b) Annealing involves binding of the primers at between 40°C - 60°C.
(c) Primer extension occurs at 68°C - 72°C.
(d) A complete reaction can be incubated at 4°C prior to analysis of the products
(e) All of the above.
(e) All of the above.
In sequential order, what are the three steps of PCR?
(a) Anneal primers, Extend DNA, Denature DNA
(b) Denature DNA, Extend DNA, Anneal primers
(c) Denature DNA, Anneal Primers, Extend DNA
(d) Extend DNA, Anneal Primers, Denature DNA
(e) None
(c) Denature DNA, Anneal Primers, Extend DNA
PCR requires all of the following except ________.
(a) primer
(b) ligase
(c) DNA polymerase
(d) DNA of interest
(e) Deoxyribonucleotides
(b) ligase
You decide to use PCR to determine if you have cystic fibrosis, a genetic disorder. You then decide to also check if you are at risk of developing Alzheimer’s, another genetic disorder. What PCR “ingredient” would be different in these two tests?
(a) Template
(b) Taq polymerase
(c) Nucleotides
(d) Primers
(e) A and B
(d) Primers
The basic requirement of PCR reaction includes
(a) DNA segment to be amplified
(b) Two oligonucleotides to be amplified
(c) A heat stable DNA polymerase
(d) DNA Helicase
(e) A, B and C
(e) A, B and C
PCR reaction involves:
(a) target DNA
(b) one primer only
(c) thermolabile DNA polymerase
(d) nucleotides
(e) all of the above
(a) target DNA
(d) nucleotides
Which of the following is not required during polymerase chain reactions?
(a) primers
(b) Taq DNA polymerase
(c) divalent cations
(d) ddNTPs
(e) dNTPs
(d) ddNTPs
