First pages of Bac Lab

Polymerase Chain Reaction (PCR)

• Definition: PCR stands for Polymerase Chain Reaction.

• Purpose: The primary purpose of PCR is to amplify DNA, allowing many copies of a specific DNA sequence to be made.

Process of PCR

• Diagram Summary:

  • Denaturation: Heating the DNA to separate its strands.

  • Annealing: Cooling down to allow primers to bind to the target sequences.

  • Extension: DNA polymerase extends the primers to form new DNA strands.

Master Mix Components

• Contents:

  • Water

  • Buffer

  • Four nucleotides (adenine, cytosine, guanine, thymine)

  • Oligonucleotides DNA primers

  • Heat-stable DNA polymerase

  • dNTP’s(deoxyribose nitrogenous bases triphosphate)

Primers

• Definition: Primers are short sequences of DNA that serve as a starting point for DNA synthesis.

• Function: In PCR, primers bind to specific sequences; for this lab, they bind with the 16S rRNA gene. They are added to imitate the natural DNA replication process.

DNA Synthesis Post-Priming

• Once primers are bound, DNA polymerase extends the DNA strand from the 5' end to the 3' end.

Highly Conserved Regions

• Meaning: Highly conserved regions refer to parts of a gene that exhibit little variation across different species.

• Importance: In the context of this lab, they are crucial for copying DNA from various bacterial species, enabling accurate identification.

Sample Preparation (Part 1)

• Lab Role: As a pathology lab technician, the task is to identify a bacterial sample received from a clinician.

• DNA Extraction:

  • Initial Step: Dissolving the cell wall using a digestive buffer containing proteolytic enzymes.

  • Wire Ring Purpose: Used to obtain bacterial colonies from a culture dish.

• Proteolytic Enzymes Use: Necessary to break down the cell wall to access the DNA.

• Inactivation: Proteolytic enzymes are inactivated by heating the sample in a water bath at 100°C to halt their action.

• Centrifugation: Spin the sample in a centrifuge to separate unwanted debris from the DNA solution.

Purpose of the Virtual Lab

• Overall Aim: The virtual lab aims to teach students the techniques for identifying different types of bacteria based on DNA sequences.

• Basic Steps of the Lab:

  1. Isolating bacterial samples.

  2. Copying DNA sequences through PCR.

  3. Analyzing the resulting DNA sequences.

  4. Identifying the types of bacteria based on the sequenced DNA.

16S rDNA Information

• Definition: 16S rDNA is a specific piece of DNA that codes for the small subunit of ribosomal RNA, used for identifying bacterial species.

• Identification Method: Compare the sequence obtained from the sample to a database to find matches and identify the bacterial species.