18 Questions
What is the primary function of primers in PCR?
To selectively amplify particular DNA fragments
What is the typical length of primers used in PCR?
18-25 nucleotides
What is the purpose of the annealing step in PCR?
To allow primers to anneal to their complementary sequences
What is the expected outcome of PCR when using specific primers?
Amplification of the target DNA sequence
What is the purpose of using specific primers in PCR?
To selectively amplify particular DNA fragments
What is the primary function of the primers in PCR?
To bind to the DNA template strands and initiate DNA synthesis
What is the expected size of the amplicon in E. coli isolates?
147-kp
What is the purpose of the negative control in the PCR reaction?
To verify the absence of DNA contamination in the reagents
What is the role of the 5x Ready mix in the PCR reaction?
To provide the necessary components for DNA synthesis, including dNTPs, DNA polymerase, and MgCl2
What is the purpose of the centrifugation step in the protocol?
To separate the DNA from the pellet
What is the main reason for using primers with specific sequences in PCR?
To initiate DNA synthesis at a specific region
What is the purpose of the NTC (no template control) in the PCR reaction?
To verify the presence of DNA contamination in the reagents
What is the primary purpose of using accurate pipettes in DNA extraction and PCR?
To ensure accurate results by preventing contamination
What is the estimated annual number of reported human illnesses caused by Shiga-toxin producing E. coli in the United States?
73,000
What is the primary role of E. coli in relation to fecal contamination?
It is an indicator organism for fecal contamination
What is the purpose of optimizing PCR conditions, such as primer concentrations and annealing temperatures?
To enhance the efficiency and specificity of PCR reactions
What is the primary mechanism of transmission of pathogenic E. coli strains?
Incorrectly prepared food items and contaminated water sources
What is the primary function of primers in PCR reactions?
To bind specifically to the target DNA sequence
Study Notes
E. coli Culture and DNA Extraction
- E. coli isolates are cultured on Nutrient agar.
- A loopful of E. coli or a toothpick is used to suspend a colony in 200μl of nuclease-free water in a 200μl strip tube.
- The strip is then placed in a thermocycler (PCR machine) for 15 minutes at 98°C.
- The resulting lysate contains DNA, which is transferred to a new clean tube, and the pellet is discarded.
PCR Reaction Recipe
- The PCR reaction recipe includes 1X Ready mix (dNTPs, DNA Polymerase I, MgCl2), primers F and R, DNA, and nuclease-free water.
- The total reaction volume is 25μl.
Controls and Primers in PCR
- Primers are crucial in PCR, initiating DNA synthesis by DNA polymerase.
- They provide specificity, ensuring DNA synthesis only at the desired target region.
- Primers are short oligonucleotides (18-25 nucleotides) that are stable and less prone to nonspecific binding.
PCR Program
- The PCR program includes an annealing step, where the temperature is lowered to allow primers to bind to their complementary sequences on the DNA template.
- The program also includes denaturation, extension, and amplification steps.
Learning Outcomes
- Understanding of DNA extraction techniques.
- Awareness of good laboratory practices to prevent contamination and ensure accurate results.
- Understanding of PCR principles, including denaturation, annealing, and extension steps.
- Understanding of optimizing PCR conditions to enhance efficiency and specificity.
E. coli and PCR Application
- E. coli is a Gram-negative enteric species with both commensal and pathogenic members.
- Pathogenic E. coli strains, such as STEC, are a major public health concern.
- PCR can be used to identify and detect E. coli in diarrheagenic stool and water samples.
This quiz covers the fundamentals of DNA extraction from bacteria and the identification of E. coli in diarrheagenic stool using Polymerase Chain Reaction (PCR). It assesses understanding of DNA extraction techniques, laboratory practices, and the principles of PCR.
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