Recombinant DNA Technology

Recombinant DNA Technology

Recombinant DNA technology refers to methods used to isolate, modify, and clone genes by inserting them into vectors for replication and study.

Gene cloning

The process of isolating a specific gene of interest and inserting it into a vector so it can be replicated and studied.

Clone

Something that is genetically identical to another.

Vector

A DNA molecule used to carry foreign DNA into a host cell and replicate it.

Common cloning vector

A bacterial plasmid.

Plasmid

A small, circular DNA molecule separate from the bacterial chromosome that can replicate independently.

Origin of replication

A DNA sequence that allows a plasmid to replicate independently inside a host cell.

Selectable marker

A gene on a plasmid that allows identification of cells that have taken up the plasmid, often antibiotic resistance.

Restriction enzyme

An enzyme that cuts DNA at specific recognition sequences.

Restriction site

A short, specific DNA sequence recognized and cut by a restriction enzyme.

Palindromic sequence

A DNA sequence that reads the same forwards and backwards on complementary strands.

Sticky ends

Overhanging DNA ends produced by staggered cuts from restriction enzymes.

Blunt ends

DNA ends produced by restriction enzymes that cut straight across both strands.

Example of a restriction enzyme producing sticky ends

EcoRI.

Example of a restriction enzyme producing blunt ends

AluI.

PCR (Polymerase Chain Reaction)

A technique used to amplify a specific DNA sequence.

When PCR is used in cloning

When the gene of interest is well studied and under 5 kb.

Alternative to PCR for gene isolation

Digesting genomic DNA with restriction enzymes.

Ligation

The joining of DNA fragments together using DNA ligase.

DNA ligase

An enzyme that forms phosphodiester bonds between DNA fragments.

Phosphodiester bond

The bond that links nucleotides together in DNA.

Recombinant DNA

DNA formed by combining DNA from different sources.

Transformation

The process of introducing a plasmid into a bacterial cell.

Competent cell

A bacterial cell capable of taking up foreign DNA.

Method used to induce transformation

CaCl₂ treatment followed by heat shock at 42 °C.

Purpose of heat shock

To increase permeability of the bacterial membrane so plasmid DNA can enter.

Antibiotic resistance gene

A selectable marker used to identify bacteria that have taken up the plasmid.

How transformation success is tested

By growing bacteria on agar containing antibiotic.

Clone screening

The process of identifying bacteria that contain the correct recombinant plasmid.

LacZ gene

A gene encoding β galactosidase enzyme that breaks down lactose into glucose and galactose

IPTG

A molecule that induces expression of the LacZ gene.

Blue colony

Indicates functional LacZ gene and no inserted gene of interest.

White colony

Indicates disrupted LacZ gene due to successful insertion of foreign DNA.

Plasmid pUC18/19

An example of a commonly used cloning plasmid.

Why vectors must have unique restriction sites

To allow insertion of foreign DNA at a single, specific location.

Two naturally occurring DNA molecules used as vectors

Plasmids and bacteriophages.

Main stages of gene cloning

Isolation of gene, vector selection, ligation, transformation, clone screening.