30 Questions
What is the primary function of thermostable DNA polymerase in PCR?
To synthesize new strands of DNA
What is the purpose of the initialization step in PCR?
To activate heat-labile DNA polymerases
What is the role of magnesium in PCR?
To act as a co-factor for DNA polymerase
What is the purpose of the denaturation step in PCR?
To denature the DNA template
What is the typical number of temperature cycles in a PCR reaction?
25-40
What is the name of the thermophilic bacterium from which Taq DNA polymerase was originally isolated?
Thermus aquaticus
What is the purpose of the PCR buffer in PCR?
To create optimal conditions for Taq DNA polymerase activity
What are dNTPs used for in PCR?
To provide a substrate for DNA polymerase activity
What is the temperature range for the annealing step in PCR?
50-65°C
What is the purpose of the final elongation step?
To ensure complete extension of remaining single-stranded DNA
What is the function of the thermostable DNA polymerase during annealing?
To extend the primers as soon as they anneal to the template
What is the optimal temperature for Taq polymerase activity?
72-80°C
What is the direction of DNA synthesis during the extension step?
5' to 3'
What is the purpose of gel electrophoresis in PCR?
To detect the amplified product
What happens to the single strands of the template DNA during the annealing step?
They remain single-stranded and do not reanneal
What is the outcome if the target sequence is not present in the original DNA sample?
No band is visible on the gel
What is the temperature in the denaturation step of a typical PCR thermal cycle?
94°C
What is the primary application of PCR in forensic science?
Identification of criminal
What is the purpose of the final elongation step in PCR?
To ensure complete extension of the DNA strands
What is the advantage of PCR in detecting microorganisms?
It can detect microorganisms that are difficult to culture
What is the purpose of PCR in medicine?
To detect pathogens
What is the primary application of PCR in evolutionary studies?
Phylogenetic analysis
What is the primary function of the thermal cycler in PCR?
To heat and cool the reaction tubes to achieve the required temperatures
What is the purpose of the forward and reverse primers in PCR?
To initiate DNA synthesis and specify the amplification to the target DNA segment
What is the minimum amount of DNA required for PCR to work?
A single copy of the DNA sequence
Who developed the Polymerase Chain Reaction (PCR) technique?
Kary Mullis
What is the function of the template DNA in PCR?
To carry the DNA segment or target you wish to amplify
What is the importance of free nuclease water in PCR?
It must be present for the reaction to work
What is the end result of the PCR process?
Thousands to millions of copies of the DNA sequence
What is the name of the device used to heat and cool the reaction tubes in PCR?
Thermal cycler
Study Notes
The Polymerase Chain Reaction (PCR)
- A scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Requirements of PCR Technology
- The thermal cycler: heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.
- Template DNA: carries the DNA segment or target you wish to amplify.
- Free nuclease water: must be present for the reaction to work.
- Forward and Reverse Primers: short, single-stranded pieces of DNA that anneal to their complementary sequence on the template, providing initiation sites for DNA synthesis.
- Thermostable DNA polymerase (e.g., Taq polymerase): synthesizes new strands of DNA by adding nucleotides onto the end of an annealed primer.
- dNTPs: the four nucleotides used by DNA polymerase to extend an annealed primer (building blocks).
- Magnesium: required for DNA polymerase activity (co-factor).
- PCR buffer: necessary to create optimal conditions for Taq DNA polymerase activity.
PCR Procedure
- A series of 25-40 repeated temperature changes (cycles), each cycle consisting of:
- Denaturation: heating the reaction to 94-97°C for 20-30 seconds to melt DNA.
- Annealing: cooling the reaction to 50-65°C for 15-60 seconds to allow primers to bind to the template.
- Extension/Elongation: heating the reaction to 72-80°C for extension time (dependant on DNA polymerase and DNA fragment length) to synthesize new DNA strands.
- Final Elongation (optional): a single step at 70-74°C for 5-15 minutes to ensure full extension of any remaining single-stranded DNA.
Applications of PCR
- Medicine: enables early diagnosis of malignant diseases.
- Classification of organisms.
- Mutation detection.
- Detection of pathogens.
- Gene therapy.
- Forensic science: important for the identification of criminals.
- Evolutionary studies: plays an important role in phylogenetic analysis.
- Analysis of ancient DNA from fossils.
- Mapping the human (and other species) genome.
- Isolation of a particular gene of interest from a tissue sample.
- Generation of probes: large amounts of probes can be synthesized by this technique.
- Production of DNA for sequencing.
- Diagnosis of monogenic diseases (single gene disorders).
- Detection of microorganisms: especially of organisms and viruses that are difficult to culture or take a long time.
- Detection of microbial genes responsible for some aspect of pathogenesis or antibiotic resistance.
- Crucial forensic evidence may often be present in very small quantities.
This quiz covers the polymerase chain reaction (PCR) technique in molecular biology, which amplifies specific DNA sequences for further study.
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