Molecular Cloning Overview

Molecular Cloning

This course is part of the PG Diploma in Biopharmaceutical Science as conducted by Dr. Catherine Hayes, and the session took place on October 25. The course covers the fundamental techniques and applications of molecular cloning in the field of biopharmaceutical science.

What is Cloning?

Cloning refers to a scientific technique aimed at locating, isolating, preparing, and studying small segments of DNA derived from much larger chromosomes. The term "clone" denotes an identical copy.

Reference: Chapter 9, Lehninger 5th Edition.

DNA Cloning Process

DNA cloning involves several key steps, which include:
- Separating a specific gene or DNA sequence from a larger chromosome.
- Attaching the isolated sequence to a small molecule of carrier DNA known as a vector.
- Introducing both the carrier DNA (vector) and the target DNA sequence into a host cell.
- Replicating the modified DNA thousands or millions of times through an increase in host cell proliferation and subsequent creation of multiple copies of the cloned DNA in each cell.

Why Clone DNA?

Cloning is essential for various purposes, notably:

  • To over-express a specific protein in a host cell, one must isolate, amplify, and insert the gene sequence into the host cell. This process allows for further transcription and translation into an amino acid sequence.

  • Cloning techniques form the foundation for genomic studies and proteomics, focusing on the exploration of genes and proteins at the scale of whole cells or organisms.

  • The capability of producing recombinant proteins and biopharmaceutical products fundamentally relies on cloning techniques.

Fathers of Cloning

The field of cloning owes part of its advancement to the pioneering work of:

  • Paul Berg

  • Herbert Boyer

  • Stanley N. Cohen

Cloning Overview

The cloning process can be divided into several critical steps:

  1. Isolate: Isolate the DNA from the original cell source.

  2. Amplify: Use Polymerase Chain Reaction (PCR) to amplify the DNA sequence of interest.

  3. Choose: Select a small self-replicating DNA molecule known as a cloning vector, typically a bacterial plasmid or viral DNA.

  4. Join: Covalently bond the two DNA fragments using DNA ligase, creating recombinant DNA.

  5. Insert: Introduce the recombinant DNA into the chosen host cell (e.g., transformation in E. coli), which provides the machinery to replicate this DNA.

  6. Select: Identify and select host cells that contain recombinant DNA, often facilitated by antibiotic resistance selection (details will be elaborated later).

Cloning Vectors

Cloning vectors are defined as any DNA molecule that can self-replicate inside a cell and is utilized for carrying cloned genes or DNA segments. The most common types of cloning vectors include:

  • Plasmids

  • Modified viruses

  • Artificial chromosomes

Plasmid Vectors

  • Definition: Plasmids are small, closed-circular DNA molecules found naturally in many bacteria, in addition to their chromosomal DNA. They typically consist of less than 30 genes and can range in size from 5 kb to 40 kb.

  • Characteristics:

    • Plasmids can replicate independently of the bacterial chromosome due to their own origin of replication (ori).

    • They can be categorized into single-copy (one copy per host cell) and multicopy plasmids (40 or more copies per host cell).

    • Some plasmids can integrate into the bacterial chromosome (known as episomes).

    • Although not essential for bacterial growth and reproduction, plasmids may provide a selective advantage, such as drug resistance.

Plasmid as Vector

A plasmid becomes a vector when it is synthetically modified by adding or removing specific genes or DNA sequences. Critical components of a plasmid vector include:

  • An origin of replication (ori).

  • A selection marker, typically an antibiotic resistance gene.

  • A multiple cloning site that permits the addition of more DNA, such as genes.
    The genetic configuration of a vector dictates whether it serves as a simple cloning vector or an expression vector.

Cloning Vector Characteristics

The essential characteristics of cloning vectors are:

  • Origin of replication: A designated point on the circular DNA construct where replication begins.

  • Selectable marker: Such as an antibiotic resistance gene, which confers survival capabilities to E. coli cells harboring the vector in selective media.

  • LacZ gene: Utilized for blue/white screening, whereby disruption of this gene indicates successful cloning.

  • Specific restriction sites: These are locations on the vector recognized by various restriction endonucleases that cut the DNA, allowing for the insertion of the gene of interest.

Examples of Classical Cloning Vectors

  1. pBR322: An early cloning vector characterized by an origin of replication, two antibiotic resistance cassettes (tetR for tetracycline and ampR for ampicillin), and several cloning sites that allow the entry of target genes. It is relatively small (4361 bp), facilitating entry into host cells.

  2. pUC18: This vector also possesses an origin of replication, a single antibiotic resistance gene (ampR), and a multiple cloning site located within the lacZ gene, essential for blue/white screening. The plasmid size is 2686 bp.

Simple Restriction Cloning Example

The process of simple restriction cloning with a plasmid vector involves several key steps:

  • Restriction of DNA: Complementary “sticky” ends are created on both the vector and the DNA to be inserted.

  • Mixing: The vector and the DNA of interest are combined.

  • Ligation: The ligase enzyme is added to recombine these two DNA fragments effectively.

Example of Cloning Experiment

A practical example highlighted is the cloning of an insulin gene into pUC18. The insulin gene is amplified using specific primers:

  • Forward Primer: 5’ gggccAAGTCCATGGCCCTGTGG 3’

  • Reverse Primer: 5’ ggttaaGGATTCCTAGTTGCAGTAG 3’
    These primers create an insulin gene with specified overhangs, suitable for insertion into the plasmid.

PCR Amplification and Restriction Digestion

In this process, PCR amplifies the insulin gene alongside generating “overhangs” that feature restriction sites, enabling the precise insertion into the cloning vector. After this, restriction enzymes HindIII and BamHI are used for digestion, producing sticky ends required for ligation.

Ligation Process

During ligation, the complementary sticky ends of the insert and the plasmid attract each other. Following incubation with the ligase enzyme, the nicks in the DNA are sealed, establishing a phosphodiester bond between the nucleotides of the insert and the vector, yielding a new plasmid.

Other Cloning Techniques

Various alternative cloning methods exist, including:

  • Golden Gate Cloning

  • Topo Cloning

  • Sequence and Ligation Independent Cloning

  • Gateway Cloning

  • Gibson Cloning

Vector Reference Sequence and Data

An example dataset of pUC18 includes notable features and restriction sites relevant for cloning (e.g., AmpR, lacZ). This dataset forms a reference for vector construction and application in cloning experiments.

This extensive outline reflects a comprehensive examination of molecular cloning techniques and their significance in biopharmaceutical science as presented in the lecture. Each concept and process is derived from presented details and definitions, allowing students to fully grasp the cloning methods and their applications within the field.