mitochondrial genome. This can be done using three water baths
Introduction Soon after the rediscovery of Mendel’s Laws, patterns of inheritance were observed that suggested the existence of extrachromosomal genes in plants. It was not until 1962, however, that definitive proof that chloroplasts contain DNA was provided. In most species, chloroplast DNA (ctDNA) is inherited through the maternal line and has a low mutation rate. It can therefore provide important clues about evolution. Between 10–100 chloroplasts are found in each photosynthesizing cell, and each chloroplast contains 50–100 copies of the ctDNA.
Some 10–20% of a plant’s total DNA is found in the chloroplasts. Like that of plasmids and mitochondria, ctDNA is circular; typically it is 120–150 kb in length, encoding ~80 proteins. Many of the proteins that chloroplasts require to function are, however, encoded in the nuclear DNA. These include, for example, the enzymes needed for replication of the ctDNA. To date, the ctDNA of about 25 species has been fully sequenced. Aim School students are able to prepare crude extracts of DNA from a variety of plant sources, using simple procedures [1].
Since the advent of pre-aliquotted restriction enzymes, inexpensive electrophoresis systems and safe staining methods, it has also become common for school students to carry out restriction digests and gel electrophoresis of lambda DNA [2, 3]. Unfortunately, when students isolate nuclear DNA from plants and try to obtain visible bands using gel electrophoresis after a restriction digest, the results are often disappointing. This is because of the great length and numerous restriction sites found in plants’ nuclear DNA.
The many fragments formed produce a ‘smear’ on the gel rather than distinctive bands. This paper describes a simple but effective method for the isolation of relatively pure ctDNA. In contrast to plant nuclear DNA, ctDNA is small and has few restriction sites. Consequently it is possible to digest this DNA to produce clear bands following electrophoresis. This technique presents ample opportunities for open-ended investigations. The isolation method described here is a simplified version of that developed by Bookjans et al [4]. This method relies upon a highsalt extraction buffer, which minimises nuclear contamination — unlike similar extraction media that contain sucrose as an osmoticum for keeping chloroplasts intact.