Everything from the 5 lessons of this topic on a few pages: the key idea of each lesson, the diagram to remember, the points to learn and the traps examiners set. Read it, cover it, test yourself.
Recombinant DNA is DNA made by joining together DNA from two different sources, often from different species. Genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism; often a gene is transferred into another organism so that it is expressed there. The gene can be extracted from donor DNA with restriction endonucleases, made from mRNA with reverse transcriptase and DNA polymerase, or synthesised chemically from nucleotides. A plasmid cut with the same restriction endonuclease has complementary sticky ends; DNA ligase seals the gene into it. A promoter that the host's RNA polymerase recognises is transferred with the gene so that it is transcribed, and a marker gene coding for a fluorescent protein such as GFP shows which cells are expressing the new DNA.
Reverse transcriptase makes DNA from an RNA template (the reverse of transcription). It does not make mRNA from DNA, and it is not the enzyme that cuts DNA.
Seeing the gene in the cell is not enough: the marker shows that the inserted DNA is being expressed (transcribed and translated), because the green colour only appears if a fluorescent protein has been made.
The polymerase chain reaction (PCR) clones and amplifies a chosen section of DNA. A tube holds the template DNA, two primers, free DNA nucleotides, Taq polymerase and buffer, and a thermal cycler repeats three steps: denaturation at about 95 °C (hydrogen bonds break, strands separate), annealing at about 55 °C (primers bind to complementary sequences at each end of the target) and extension at about 72 °C (Taq polymerase adds nucleotides to the primers). Every cycle doubles the DNA, so n cycles give 2n copies. Taq polymerase comes from a bacterium of hot springs and is thermostable, so it survives 95 °C. In gel electrophoresis, DNA is loaded into wells in an agarose gel; its phosphate groups make it negatively charged, so it moves towards the anode, and shorter fragments move further through the mesh of the gel.
Primers are DNA, not RNA, in PCR, and there are two of them: one binds to each strand at opposite ends of the target, so the section between them is what gets copied.
Distance travelled is not proportional to length: it falls roughly with the logarithm of the fragment length, so always estimate sizes by comparing with a ladder on the same gel.
A microarray is a small slide carrying thousands of spots, each holding many copies of a single-stranded DNA probe for one gene or sequence. To study gene expression, mRNA is extracted from two samples, converted to cDNA by reverse transcriptase, and labelled with different fluorescent dyes. The cDNA hybridises to complementary probes, unbound DNA is washed off, and a scanner records the colour and brightness of every spot: which genes are expressed in each sample, and how strongly. To analyse genomes, labelled fragments of genomic DNA are used instead, showing which genes or alleles two genomes share. Bioinformatics databases store nucleotide sequences of genes and genomes, amino acid sequences of proteins and protein structures, so scientists anywhere can search, compare and use them.
A dark spot in an expression study does not mean the gene is missing from the cells. It means the gene was not being transcribed, so there was no mRNA, and therefore no cDNA, to bind.
Gene editing is a form of genetic engineering in which DNA is inserted, deleted or replaced at a specific site in the genome, for example using a guide RNA to lead a nuclease to the target. Recombinant human proteins such as insulin (diabetes), factor VIII (haemophilia) and adenosine deaminase (one form of SCID) are identical to the human proteins, made in large amounts and free from the infection risks of animal or donor sources. Genetic screening tests DNA for particular alleles, such as harmful BRCA1 and BRCA2 alleles (breast cancer), the Huntington's disease allele and cystic fibrosis alleles, so people can make informed decisions and start treatment early. Gene therapy adds a working allele to a patient's cells using a vector, as in treatments for SCID and inherited eye diseases. All of these raise social and ethical questions about consent, privacy, discrimination, cost, safety and the limits of what should be changed.
Bacteria cannot add sugar groups or modify proteins the way human cells do, so some recombinant human proteins, such as factor VIII, are made in cultured mammalian cells instead.
Genetic engineering can help meet the global demand for food by improving the quality and productivity of farmed animals and crops. GM salmon carry a growth hormone gene from Chinook salmon with a promoter from ocean pout that keeps it switched on all year, so they reach market size in about half the time on less feed. Herbicide-resistant soybean carries a bacterial gene for a form of the enzyme EPSPS that is not inhibited by glyphosate, so fields can be sprayed to kill weeds without harming the crop. Insect-resistant (Bt) cotton carries a gene from Bacillus thuringiensis for a Bt toxin that kills caterpillars that eat it, so less insecticide is needed. The ethical and social implications include benefits to yield and farmers' incomes, and concerns about gene flow, effects on other species, resistance in weeds and pests, the cost of seed and control by large companies, animal welfare, labelling and consumer choice.
Herbicide-resistant crops do not kill weeds themselves. They survive the herbicide, which allows the farmer to spray it onto the growing crop. Bt crops are the ones that make their own insecticide.