Cycle 10: DNA Technologies I

Fundraiser Announcement

  • Professor Maxwell will be pied for Make A Wish Canada Foundation
  • Event details:
    • Date: March 19
    • Time: 1:15 PM - 1:40 PM
    • Location: UCC Atrium

Overview of Lecture Topics

  • Today's focus: PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription Polymerase Chain Reaction)
  • Next lecture: CRISPR

Importance of Understanding Natural Systems

  • Most bioscience techniques stem from natural processes
  • PCR is derived from DNA synthesis
    • Required reading: DNA Synthesis in the textbook

What is PCR?

  • Definition: Polymerase Chain Reaction for amplifying a specific region of DNA
  • Purpose:
    • Amplify small quantities of DNA for various analyses
    • Analogy: Need sufficient DNA to perform experimental work (e.g., baking a cake with enough flour)
  • PCR cannot amplify entire genomes, only specific target regions.

Origins and Impact of PCR Technique

  • Developed by Kary Mullis and Michael Smith, Nobel Prize in 1993
  • Revolutionized bioscience and forensic science
  • Significance over CRISPR: PCR is focused on amplification, while CRISPR deals with genome editing, which has older techniques like TALENs.

Understanding RT-PCR

  • RT-PCR stands for Reverse Transcription PCR
  • Allows for quantification of gene expression by converting mRNA to cDNA.
  • Comparison to northern blot for gene expression analysis.
  • Key applications:
    • Gene expression studies
    • Cloning
    • Disease diagnosis and paternity testing

Applications of PCR and RT-PCR

  • PCR Applications:
    • DNA profiling (e.g., forensic investigations)
  • RT-PCR Applications:
    • Production of human insulin using bacteria
    • Quantifying mRNA from cells

Technique of PCR

  1. Denaturation (95°C): DNA strands separate.
  2. Annealing (55°C): Primers bind to specific sequences.
  3. Extension (72°C): DNA polymerase synthesizes new DNA strands.
  • Key components: Taq polymerase, dNTPs, and magnesium chloride.
  • Taq polymerase sourced from thermophiles, allowing it to withstand high temperatures.

Multiplex PCR

  • Multiple sets of primers can be used in one PCR tube to amplify multiple regions simultaneously.
  • Useful in DNA profiling to identify relationships or match DNA samples in forensic cases.

DNA Profiling and STRs

  • Short Tandem Repeats (STRs):
    • Sequences between genes, used to differentiate individuals due to high polymorphism and low mutation rates.
    • Example: CODIS system identifies specific STR loci.
  • Analysis of multiple STRs increases the accuracy of DNA matches:
    • 13 STRs: one in hundreds of trillions chance of false match.

Electropherogram for DNA Analysis

  • Results from a PCR visualized via gel electrophoresis or electropherograms, showing distinct peaks representing different STR lengths.
  • Importance of controls to validate results.

Reverse Transcription Steps in RT-PCR

  1. Isolation of RNA phase: Focus on mRNA, which contains coding sequences only.
  2. Reverse Transcription: Convert mRNA to cDNA using reverse transcriptase, primer specific to poly A tail.

Production of Human Insulin Using RT-PCR

  • Process involves:
    • Isolating mRNA from pancreatic cells
    • Reverse transcription to create cDNA
    • Amplifying cDNA using PCR
    • Inserting cDNA into plasmids for bacterial expression, leading to insulin production without intronic sequences.
  • Result: Bacteria produce functional human insulin.

Summary of PCR and RT-PCR

  • PCR is essential for amplifying DNA; RT-PCR enables the study of gene expression through cDNA synthesis. Both techniques are crucial in research, diagnostics, and forensic science.