Recombinant DNA Technology

Lecture Overview

  • Molecular Biology & Biochemistry Lecture 5: Recombinant DNA Technology

  • Instructor: Louise Jones (louise.jones@york.ac.uk)

  • Lecture Location: CORI-HR #R3101

Learning Outcomes

  • LO1: Apply knowledge of DNA replication to understand PCR and DNA sequencing principles.

  • LO2: Describe how recombinant DNA molecules can be created using restriction enzymes and DNA ligase.

  • LO3: Understand how plasmids can be used to clone pieces of DNA.

DNA Manipulations

  • DNA can be:

    • Amplified

    • Sequenced

    • Cut and rejoined into recombinant molecules

    • Cloned in plasmid vectors

Polymerase Chain Reaction (PCR)

  • Specific DNA Amplification: Allows amplification of specific DNA sequences.

  • Inventor: Kary Mullis, awarded 1993 Nobel Prize in Chemistry for developing PCR method.

Requirements for PCR

  • Based on DNA replication:

    • Deoxynucleoside triphosphates (dNTPs)

    • Template DNA

    • A 3’ end for nucleotide addition

    • DNA polymerase enzyme

    • Energy source

Primers in PCR

  • Essential for targeting specific regions of DNA.

  • Short single-stranded DNA (~21 nucleotides) that annel with template strands at the edges of regions to be amplified.

  • Types of primers:

    • Forward Primer

    • Reverse Primer

PCR Process

Stages of a PCR Cycle

  1. Denaturation (96°C): DNA strands separate.

  2. Annealing (~55°C): Primers bind to the template.

  3. Extension (72°C): DNA polymerase synthesizes new DNA strands.

  • Each cycle doubles the amount of DNA.

  • Thermus aquaticus provides Taq Polymerase, a thermostable enzyme for the extension phase.

Visualization of PCR Products

  • PCR products can be analyzed using gel electrophoresis:

    • DNA is negatively charged and migrates towards the positive electrode.

    • Smaller DNA fragments move faster through an agarose gel.

Applications of PCR

  • Forensics

  • Diagnostics

  • Genotyping

  • Generating DNA fragments for recombinant molecules

DNA Sequencing

  • Involves polymerization similar to PCR.

  • Sanger Sequencing: Chain termination method requiring:

    • DNA template

    • Primer

    • DNA polymerase

    • Regular and chain-terminating nucleotides.

Chain Elongation and Terminology

  • The 3’ OH group is crucial for DNA polymerization.

  • Dideoxynucleotides lack a 3’ OH and terminate chain extension, generating fragments of differing lengths based on their termination points.

Capillary Electrophoresis in Sequencing

  • DNA fragments of varying lengths are separated, with the shortest fragments traveling fastest.

  • Laser detection of these fragments allows identification of the DNA sequence.

Restriction Enzymes

  • These enzymes recognize specific DNA sequences and cleave the phosphodiester backbone, creating "sticky" or "blunt" ends:

    • Example: EcoRI cuts at 5′-GAATTC-3′, producing sticky ends.

  • Different enzymes produce different end types:

    • EcoRI: 5’ sticky ends

    • KpnI: 3’ sticky ends

    • SmaI: blunt ends.

Ligation Reactions**

  • DNA ligase facilitates the joining of DNA fragments with compatible ends, forming recombinant DNA molecules.

  • Ligation efficiency varies based on DNA end compatibility (sticky vs. blunt ends).

Plasmid Cloning**

  • Plasmids are circular DNA molecules that can replicate independently in bacteria and can be modified to carry foreign DNA.

  • Components of plasmid cloning include:

    • Origin of replication

    • Antibiotic resistance gene

    • Gene of interest (insert).

Transformation Processes

  • Transformation: Mechanism by which bacteria take up foreign DNA.

    • Requires chemical treatment/heat shock for laboratory strains like E. coli to become competent.

Selection of Transformed Bacteria

  • Bacteria can be selected based on antibiotic resistance conferred by the plasmid after transformation.

Plasmid Cloning Experiment Overview**

  • Possible outcomes post-transformation include:

    • No plasmid

    • Unligated vector

    • Growth if desired insert is present.

Identifying Recombinant Plasmids

  • Utilizes antibiotic resistance and the lacZ gene for blue/white screening:

    • Insert disrupts lacZ resulting in non-functional β-galactosidase, leading to white colonies forming in X-gal solution.

Conclusion and Further Study**

  • Recombinant DNA technology is an evolution of DNA replication knowledge.

  • DNA sequencing, PCR, and cloning methods are integral to advanced biological studies.

  • Suggested follow-up: Review learning outcomes, participate in workshops, and read about the history and ethics of recombinant DNA technology.