Principles of genetic technology

Biology A2 · Genetic technology. The idea in plain words, the rules to remember, the mistake to avoid and a worked example, written and checked by Mega Lecture.

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The idea

Genetic engineering is the deliberate changing of genetic material to alter a characteristic of an organism. Often a gene is moved into another organism and expressed there. DNA made by joining DNA from two or more sources is called recombinant DNA.

The gene can be cut from donor DNA, made from donor mRNA or built from nucleotides. It is joined into a vector, usually a plasmid, and put into a host cell. A promoter is added so that the host transcribes the gene, and a marker gene shows which cells express it.

PCR copies a chosen piece of DNA many times. Gel electrophoresis separates DNA fragments by length. Microarrays and databases help to analyse genes and their expression.

Rules to remember

Restriction endonucleases cut DNA at specific base sequences, often leaving sticky ends; cut the gene and the plasmid with the same enzyme.
DNA ligase joins the sugar-phosphate backbones by forming phosphodiester bonds.
Reverse transcriptase makes single-stranded cDNA from mRNA; DNA polymerase then makes the second strand.
A promoter is where RNA polymerase binds; without one that the host recognises, the gene is not transcribed.
PCR cycle: denature at about 95 °C, anneal primers at about 55–65 °C, extend with Taq polymerase at about 72 °C. The amount of DNA doubles each cycle.
Taq polymerase is heat stable, so it is not denatured at 95 °C.
Gel electrophoresis: DNA is negative (phosphate groups) and moves to the anode; shorter fragments move further.

Common mistake

Students say DNA ligase joins the bases, or forms hydrogen bonds. Hydrogen bonds between sticky ends form by themselves; ligase makes the phosphodiester bonds in the backbone.

Worked example

A linear piece of DNA is 6000 base pairs long. A restriction enzyme cuts it at positions 1500 and 4000. Which fragments are produced, and which moves furthest in gel electrophoresis?

Two cuts in a linear molecule give three fragments.
Lengths: 1500 − 0 = 1500 bp, 4000 − 1500 = 2500 bp, 6000 − 4000 = 2000 bp.
Check: 1500 + 2500 + 2000 = 6000 bp.
The shortest fragment passes through the gel most easily.

Answer: Fragments of 1500, 2000 and 2500 bp; the 1500 bp fragment moves furthest towards the anode.

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