Recombinant DNA, Cloning and PCR

A desired fragment can be cut from DNA using restriction endonucleases, made synthetically from a specified sequence, or produced as complementary DNA using reverse transcriptase and an RNA template. Mature mRNA provides an intron-free coding message, which can be useful when expression is planned in a bacterial host that does not perform the same eukaryotic splicing.

Restriction enzymes recognise particular sequences and can create ends suitable for joining. DNA ligase forms covalent links in the sugar–phosphate backbone when appropriately matched fragments are assembled. Base pairing between complementary ends alone does not complete all the covalent joins.

PCR uses a DNA template, primers flanking the target region, free nucleotides and a suitable thermostable DNA polymerase. Heating separates strands, cooling allows primers to anneal to complementary sites, and an appropriate extension stage allows new DNA synthesis. Repeated cycles amplify the target.

In an ideal doubling model, copy number follows N=N0×2ᶜ for c cycles. Real efficiency and target yield depend on conditions and eventually plateau. PCR copies DNA; it does not itself translate the gene into a protein or establish that the fragment is functional.

A vector such as a plasmid can carry an inserted fragment into host cells. Appropriate promoter and terminator sequences support expression in a compatible system. Transformed host cells can be cultured to copy the vector and, when suitable regulatory and cellular conditions are present, produce the encoded product.

A marker helps identify cells with relevant vector properties, but a marker result alone may not prove that the intended insert is present, correctly oriented and expressed. Further screening and product testing can be needed. AQA does not require memorising particular named marker genes, so concentrate on the evidence each marker provides.

Shared genetic coding supports transfer of a coding sequence between organisms, but compatible regulation and processing are also needed. Applications include production of useful proteins, altered crop properties and gene-therapy approaches that supply or modify a functional sequence.

Evaluate effectiveness, expression control, unintended consequences, environmental spread, ownership, access and informed consent where relevant. Adding a functional gene copy need not remove the original allele, and a somatic intervention is not automatically inherited. Use the actual proposed mechanism and evidence rather than treating every genetic modification as the same intervention.

Try each question before opening the worked response. Use precise biological terms, connect each cause to its effect, and check units and assumptions when interpreting data.