Genetic Engineering

Genetic engineering (also called recombinant DNA technology) is the deliberate manipulation of genes to transfer them from one organism to another, producing transgenic or genetically modified (GM) organisms. OCR A-Level Biology A specification 6.1.3 (f)–(h) requires you to understand the molecular toolkit — restriction enzymes, ligases, vectors, marker genes — and the standard cloning workflow used to create GM bacteria, plants and animals.

Key Definitions:

• Recombinant DNA — DNA formed by joining DNA from two different sources.
• Restriction endonuclease — an enzyme that cuts DNA at a specific recognition sequence.
• Sticky ends — single-stranded overhangs produced by restriction enzymes that cut asymmetrically.
• DNA ligase — an enzyme that catalyses the formation of phosphodiester bonds between DNA fragments.
• Vector — a DNA molecule (typically a plasmid or virus) used to carry foreign DNA into a host cell.
• Transformation — the uptake of foreign DNA by a cell.
• Marker gene — a gene introduced alongside the gene of interest to identify successfully transformed cells.

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The Molecular Toolkit

Genetic engineering became possible in the 1970s with the discovery of two key enzymes:

1. Restriction endonucleases — discovered in bacteria, where they defend against invading phages by cutting foreign DNA. Each enzyme recognises a specific palindromic sequence (typically 4–8 bp). The famous EcoRI from E. coli recognises GAATTC and cuts between G and A, leaving 5' AATT overhangs.
2. DNA ligase — the same enzyme used in DNA replication, which seals nicks in the sugar–phosphate backbone by forming phosphodiester bonds.

Together these enzymes allow scientists to cut DNA at predictable positions and rejoin fragments from different sources.

Sticky Ends vs Blunt Ends

Some restriction enzymes (like EcoRI) cut asymmetrically, leaving sticky ends — short single-stranded overhangs that can base-pair with complementary sticky ends from any other fragment cut with the same enzyme. Others (like SmaI) cut symmetrically, producing blunt ends. Sticky ends are preferred for cloning because they hybridise specifically and increase ligation efficiency.

Enzyme	Source	Recognition sequence	Cut type
EcoRI	E. coli	GAATTC	Sticky (5' AATT)
BamHI	Bacillus amyloliquefaciens	GGATCC	Sticky (5' GATC)
HindIII	Haemophilus influenzae	AAGCTT	Sticky (5' AGCT)
SmaI	Serratia marcescens	CCCGGG	Blunt

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The Standard Cloning Workflow

flowchart TD
    A[Isolate gene of interest] --> B[Cut gene with restriction enzyme]
    C[Cut plasmid vector with same enzyme] --> D[Mix gene + vector]
    B --> D
    D --> E[DNA ligase seals phosphodiester bonds]
    E --> F[Recombinant plasmid]
    F --> G[Transform into host bacterium]
    G --> H[Select transformed cells using marker genes]
    H --> I[Culture and express protein]

Step 1: Isolate the Gene of Interest

Three main methods:

• Reverse transcription — extract the mRNA for the target gene from a cell expressing it (e.g. insulin mRNA from β-cells). Use the enzyme reverse transcriptase (from retroviruses) to make a complementary DNA (cDNA) copy. cDNA lacks introns — important when expressing eukaryotic genes in prokaryotic hosts that cannot splice them.
• Restriction digestion — cut the gene out of genomic DNA using enzymes flanking the gene.
• Gene machine — synthesise the gene from scratch using an automated DNA synthesiser. This is now routine and increasingly cheap.

Step 2: Insert the Gene into a Vector

Plasmids are the most common vectors: small, circular, double-stranded DNA molecules found naturally in bacteria. They replicate independently of the chromosome and can carry foreign DNA up to about 10 kb. Larger inserts require other vectors:

• Bacteriophages — viruses that infect bacteria; carry up to about 25 kb.
• Cosmids — hybrid plasmid/phage vectors; up to 45 kb.
• Artificial chromosomes — yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) carry hundreds of kb.