SN5 SHORT NOTE 5M ★ High-yield

Describe the tools of recombinant DNA technology and its medical applications.

Biochemistry · 1st Professional — Pre-Clinical

Definition

Recombinant DNA technology (genetic engineering) = the set of techniques by which DNA from different sources is joined and introduced into a host cell, where it can be replicated and expressed.

The Tools

Tool	Function
Restriction endonucleases	"Molecular scissors" — cut DNA at specific palindromic sequences, leaving sticky or blunt ends. Example: EcoRI
DNA ligase	"Molecular glue" — joins the fragments
Vectors	Carry the insert into the host — plasmids, bacteriophage, cosmids, BAC, YAC
Host cell	E. Coli, yeast, mammalian cells
Reverse transcriptase	Makes cDNA from mRNA
Probes	Labelled DNA used to identify a sequence

The Basic Steps

Isolate the DNA of interest → Cut both insert and vector with the same restriction enzyme → Ligate to form recombinant DNA → Transform into the host cell → Select the successful clones (antibiotic resistance, blue–white screening) → Express and harvest the product

Blotting Techniques

Technique	Detects	Probe	Use
Southern blot	DNA	Labelled DNA	Gene deletions, RFLP, DNA fingerprinting
Northern blot	RNA	Labelled DNA or RNA	Gene expression
Western blot	Protein	Antibody	Confirmatory test for HIV

CLINICAL PEARL

Mnemonic — Southern for DNA, Northern for RNA, Western for Protein: "SNoW DRoP".

Medical Applications

Therapeutic proteins — human insulin (the first, 1982), growth hormone, erythropoietin, clotting factor VIII, interferons, tPA
Vaccines — recombinant hepatitis B vaccine; HPV vaccine; mRNA vaccines
Monoclonal antibodies — rituximab, trastuzumab, infliximab
Diagnosis — gene probes, PCR, prenatal and carrier detection
Gene therapy — ADA-SCID was the first success; now used in haemophilia, spinal muscular atrophy and some retinal disease
CRISPR-Cas9 gene editing — precise correction of mutations; already licensed for sickle cell disease and β-thalassaemia

Applied Aspects

Recombinant human insulin removed the risk of allergy to animal insulin and the dependence on pancreas supply — the clearest early benefit of the technology
Recombinant factor VIII eliminated the transmission of HIV and hepatitis C that devastated the haemophilia population in the 1980s
Ethical concerns — germline editing is heritable and remains widely prohibited; questions of consent, equity of access and genetic privacy are unresolved
Gene therapy remains limited by delivery, immune responses to vectors, durability and cost

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