DNA Extraction and PCR in Bacteria Identification

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18 Questions

What is the preferred indicator organism for fecal contamination?

E.coli

What is the estimated number of annual cases of human illnesses caused by Shiga-toxin producing E. coli variants in the United States?

73,000

What is the primary mode of transmission of pathogenic E. coli strains?

Contaminated food and water sources

What is the role of DNA polymerase in PCR?

To synthesize new DNA strands

What is the purpose of optimizing PCR conditions?

To enhance PCR efficiency and specificity

What is the learning outcome of understanding the principles of PCR?

Understanding the principles of PCR

What is the main advantage of selecting specific primers in PCR?

To selectively amplify particular DNA fragments from complex mixtures of DNA

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 the primers to anneal to their complementary sequences on the DNA template

What is the result of PCR amplification of the target DNA sequence?

Millions of copies of the target region

What is the purpose of using specific primers in the experiment described?

To selectively amplify specific virulent genes in E. coli

What is the concentration of primers used in the experiment?

10 pmol

What is the purpose of the thermocycler in step 3?

To lyse the E.coli cells

What is the function of the primer in PCR?

To initiate DNA synthesis by binding to the target DNA region

What is the purpose of the negative control in the PCR reaction?

To confirm the absence of PCR inhibitors

What is the composition of the 5x Ready mix?

dNTPs, DNA polymerase, and MgCl2

Why are primers designed to be specific to the target DNA region?

To ensure that DNA synthesis initiates only at the desired target region

What is the purpose of centrifuging the mixture at 13,000 rpm for 3 minutes?

To separate the DNA from the cell debris

Study Notes

DNA Extraction and PCR for E. coli Identification

  • Escherichia coli (E. coli) is a Gram-negative enteric species with both commensal and pathogenic members.
  • Pathogenic E. coli strains, including Shiga-toxin producing variants (STEC), cause approximately 73,000 annual cases of human illnesses in the United States.
  • E. coli is the preferred indicator organism for fecal contamination due to its consistent association with fecal matter from warm-blooded animals.

DNA Extraction from Bacteria

  • E. coli isolates are cultured on Nutrient agar.
  • A loopful of E. coli is suspended in 200µl of nuclease-free water.
  • The suspension is heated at 98°C for 15 minutes, then centrifuged at 13,000 rpm for 3 minutes.
  • The lysate containing the DNA is transferred to a new tube, and the pellet is discarded.

PCR Reaction Recipe

  • The PCR reaction mixture consists of:
    • 12.5µl of 5x Ready Mix (containing dNTPs, DNA Polymerase I, and MgCl2)
    • 1µl of each primer (Forward and Reverse)
    • 2µl of DNA
    • 8.5µl of nuclease-free water
  • The total volume of the reaction mixture is 25µl.

Importance of Primers in PCR

  • Primers initiate DNA synthesis by providing a 3' OH group for DNA polymerase to extend from.
  • Primers ensure specificity by binding to specific sequences on the DNA template, allowing for selective amplification of target DNA regions.
  • Primers amplify the target DNA sequence exponentially during PCR.

PCR Program for Amplifying DNA

  • The PCR program consists of three steps:
    1. Denaturation: DNA is denatured at high temperatures.
    2. Annealing: Primers bind to their complementary sequences on the DNA template at a lower temperature.
    3. Extension: DNA polymerase extends the primers, synthesizing new DNA strands complementary to the template.

Experimental Design

  • Four groups will perform PCR using different primer pairs:
    • Group 1: HlyA-F and R
    • Group 2: eae-F and R
    • Group 3: stx2-F and R
    • Group 4: UAL754_F and UAR900_R
  • The housekeeping gene uidA (β-D-glucuronidase) will be used as a control, amplifying a 147-kb fragment in all E. coli isolates.

This quiz covers DNA extraction techniques, good laboratory practices, and the principles of Polymerase Chain Reaction (PCR) for identifying E. coli in diarrheagenic stool samples.

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