recombinent DNA

Lecture Overview

  • Course: Essential Biomedical Sciences

  • Unit: 2: Manipulating DNA

  • Lecture Number: 1

  • Topic: Recombinant DNA Technologies, Vector DNA for Cloning

  • Instructor Contact: Bradley Cobb (bcobb@rvc.ac.uk)

Learning Objectives

By the end of this lecture, students should be able to:

  • Outline the characteristics of Type II Restriction Enzymes.

  • Explain the process of cutting and re-joining DNA using restriction enzymes and DNA ligase.

  • Discuss the molecular basis of cloning DNA fragments into plasmids using bacteria and provide examples of their application.

Understanding Restriction Enzymes

Definition and Function

  • Restriction Enzymes: A type of endonuclease enzyme that cleaves the phosphodiester bonds between nucleotides in DNA.

  • These enzymes act as a bacterial immune system to protect against viral infections by recognizing and digesting foreign DNA.

  • They allow precise manipulation of DNA, which forms the basis for DNA cloning and recombinant DNA technology.

Classes of Restriction Enzymes

  • Restriction Enzymes can be categorized into four classes based on their action:

    • Type I: Cuts remote from the recognition sequence.

    • Type II: Cuts beside or within the recognition sequence.

    • Type III: Cuts near the recognition sequence.

    • Type IV: Cuts modified DNA (e.g., methylated DNA).

Focusing on Type II Restriction Enzymes

Characteristics

  • Recognize 4-8 nucleotide double-stranded DNA sequences.

  • Recognition sites are typically palindromic, allowing for symmetrical cutting within the recognition sequence.

  • An example of nomenclature is EcoRI:

    • Genus: Escherichia

    • Species: coli

    • Strain: R17

Cut Types

  • Type II restriction enzymes can create:

    • 5’ Overhangs

    • 3’ Overhangs

    • Blunt Ends

    • The ends of the DNA fragments resulting from cuts can be compatible for ligation.

DNA Ligation Process

Overview of Ligation

  • The process of ligation involves re-joining compatible sticky ends to create new phosphodiester bonds between DNA fragments. This process is facilitated by the enzyme T4 DNA Ligase.

T4 DNA Ligase Specifications

  • Source: Isolated from T4 bacteriophage.

  • Required Conditions:

    • Needs a 5’ end of DNA to be phosphorylated.

    • ATP is required for the ligation reaction.

    • Optimal temperature for ligation is 37°C, commonly performed at 14°C-25°C overnight.

The Role of Plasmids in Cloning

Features of Plasmids

  • Plasmids are circular pieces of dsDNA that are independent from the bacterial chromosome, typically ranging from 2000-10000bp in length.

  • They replicate independently within bacterial cells, with each cell containing multiple copies.

  • Key elements of plasmids include:

    • Origin of Replication (ori)

    • Antibiotic Resistance Gene (e.g., amp)

    • Multiple Cloning Site (MCS): A cluster of recognition sites for insertion of DNA fragments.

Advantages of Plasmid Cloning

  • Plasmids allow for more extensive generation of DNA than PCR, providing milligrams to grams of DNA.

  • They can amplify larger DNA fragments than PCR (approximately 12kb).

  • Cloned sequences are generally stable and can be transferred for diverse applications, enhancing the expression of genes to produce recombinant proteins.

Cloning Techniques

Cloning with One Restriction Enzyme

  • When using a single restriction enzyme (e.g., EcoRI), both the plasmid and the fragment to be cloned have matching RE sites, resulting in compatible sticky ends for ligation.

  • This can lead to the insert being cloned into the vector in two orientations, which is significant for determining the open reading frame of a protein.

Directional Cloning with Two Restriction Enzymes

  • A more precise method involves using two different restriction enzymes with different recognition sequences to generate distinct sticky ends. This ensures the fragment can only be inserted into the plasmid in one orientation, enhancing cloning accuracy.

Transformation Process

Transformation Steps

  1. Mix circular DNA (plasmid) with competent E. coli on ice for 30 min.

  2. Heat shock at 37°C for 1 minute.

  3. Add LB broth and incubate cells at 37°C for 30-45 min.

  4. Plate the transformed E. coli cells on agar plates containing antibiotic.

  • Transformed cells survive due to antibiotic resistance conferred by the plasmid; untransformed cells die.

The Outcome of Transformation

  • Individual colonies arise from a single transformed bacterium, which multiplies, allowing cloning of the plasmid. This will lead to the ability to isolate millions of plasmid copies from the culture.

Applications of Recombinant Techniques

Case Study: Recombinant Growth Hormone

  • Growth Hormone (GH) is essential for growth, metabolism, and reproduction, traditionally sourced from human cadavers, leading to safety concerns (e.g., Creutzfeldt-Jakob Disease).

  • Currently, recombinant GH is produced in bacteria by amplifying the GH open reading frame (ORF), cloning it into a plasmid, and inducing expression.

Broader Uses of Recombinant Proteins

  • Foreign proteins produced in bacteria have diverse applications including:

    • Replacement proteins for clinical use (e.g., insulin).

    • Recombinant vaccines using viral/bacterial proteins.

    • Enzymatic applications for environmental cleanup and biofuel production.

  • Challenges include proper protein folding and lack of essential post-translational modifications.

Final Notes

  • By the end of this lecture, students should grasp the fundamentals of Type II restriction enzymes, the cutting and rejoining of DNA, and the cloning processes using plasmids in bacteria, along with real-world applications of these techniques.