Going with the flow
In the mid-1970s, Ed Southern (at the Medical Research Council Mammalian Genome Unit in Edinburgh) wanted to develop a method that would pinpoint a particular gene amidst the more than a billion building blocks — or basepairs — that compose the frog Xenopus laevis genome. Scientists knew that they could chop up DNA using restriction enzymes, proteins that cut DNA at particular sequences. They could then separate the resulting pieces by loading the collection onto an agarose gel and applying an electric current. The pieces would migrate at different rates, depending on size. For organisms with large genomes, however, this procedure generated a smear of DNA because of the millions of fragments. Finding a single piece of DNA that carried a specific sequence was hopeless.
Southern realized that he could accomplish his task by brute force: carving the gel into small horizontal slabs; washing the DNA out of each gel slice; attaching every portion to a separate filter; fishing for the particular DNA with a piece of matching, radioactively tagged RNA that would bind to it; and then measuring the amount of bound radioactivity. The tedium and labor involved in such a scheme spurred Southern to think of a better way.
If he could move DNA fragments from the gel to a membrane made of nitrocellulose, which grabs and clings to DNA, he knew he could then bind radioactively labeled RNA to the trapped DNA because that method was well established. However, he needed a way to transport the DNA. During pilot experiments, he realized that the trick would be to soak the DNA fragments out of the gel by forcing liquid to flow through the gel onto the nitrocellulose; he could accomplish this task by piling dry filter paper on top of the nitrocellulose, which would draw the liquid that would carry the DNA. The transfer worked. After applying radiolabeled RNA to the membrane and washing off all of it that didn’t stick to matching DNA sequences, Southern exposed the membrane to X-ray film. This procedure generated a high-resolution picture of the DNA bands that held sequences of interest.
Suddenly, scientists could detect a segment of DNA without purifying it from the rest of the genome. Researchers quickly exploited the technique of ‘Southern blotting’ for a wide variety of purposes. In 1978, they found, for example, that people with sickle cell anemia often lack the sequence for a particular restriction enzyme near the beta globin gene. Similarly, the method uncovered mutations that are associated with other ‘diseased’ versions of genes. On a large scale, it played a crucial role in mapping the human genome. Thirty years after publication, Southern’s original article holds the record for the most highly cited paper in the Journal of Molecular Biology.
Later, others developed a method for transferring and detecting RNA (as opposed to DNA) and, as a joke, called it “northern blotting.” The name stuck. Similarly, when investigators designed a related technique for proteins, they dubbed it “western blotting.” These procedures have made a huge impact on the study of genes and proteins, and have accelerated many advances in medical science.
Southern subsequently made another momentous contribution to the field of molecular biology. He conceived the notion of performing genetic analysis using tiny arrays of short DNA sequences — called DNA chips or microarrays — and he pioneered methods for building them. Because this technology allows researchers to conduct a tremendous number of experiments in parallel, it has unlocked countless realms of inquiry that biologists could only dream of 20 years ago and has already advanced the practice of medicine. For example, the use of microarrays allows cancers of the breast and blood system to be classified, which aids diagnosis and treatment.
Mini but mighty
In the mid 1970s, scientists could group people based on proteins in the blood and other bodily fluids, but these typing schemes were inadequate. For example, the ABO blood-typing system divides humans into only four groups (A, B, AB, and O). Alec Jeffreys (at the University of Leicester) wanted to find DNA that might uniquely identify individuals — variations associated with normal differences as well as those that cause disease — and he seized on the Southern blot to aid his search. He and others showed that single basepair changes at restriction sites existed, but were insufficiently informative to act as distinctive markers.
Several research groups had noticed highly variable regions present at diverse spots in the human genome. In each case, the spans — or ‘minisatellites’ — consisted of short repeated DNA sequences; different people carried different numbers of repeats.
When Jeffreys was analyzing the human myoglobin gene for other reasons, he found a minisatellite consisting of a 33-base-pair repeat. To determine whether related sequences exist, he probed the entire genome with a piece of radioactively labeled single-stranded DNA that contained multiple copies of this sequence. It bound at several sites, four of which varied greatly from person to person, differing in length by an integral number of repeat units. The repeats differed somewhat in sequence, but all carried a common core.
Jeffreys engineered a piece of single-stranded DNA that contained multiple copies of this shared core and tagged it with radioactivity. He then used this DNA to scour the human genome for additional minisatellites, reasoning that each person’s constellation of minisatellites should identify him or her because lengths vary from individual to individual. Together, they comprise a unique genetic fingerprint. Jeffreys showed that members of a family can be distinguished — and that each offspring carries only bands from the parents — half from the mother and half from the father, except in the occasional case where a new mutation crops up.
Information transfer. In the technique that Southern devised, a solution flows through the gel and onto the nitrocellulose membrane, carrying DNA with it. Once the DNA is immobilized, the membrane is immersed in liquid that contains a radioactive DNA or RNA probe that adheres to sequences of interest. After washing away unbound probe, the membrane is placed next to X-ray film,thus generating ‘bands’ that correspond to DNA fragments that stuck to the probe.
Satellites land in the real world
Jeffreys soon applied his technique to a number of practical problems. The first such use, in 1985, involved the immigration case of a UK citizen who was returning to join his mother and siblings after a long visit to his original home of Ghana. Officials said that this boy’s passport was forged and, as a consequence, he faced deportation. The authorities thought he might be a nephew or unrelated. But DNA fingerprint analysis showed that all of the boy’s DNA bands matched either those of the mother or one of her undisputed children (and by inference, the father), and the family was reunited.
Jeffreys improved and adapted the technology so it would work on tiny amounts of forensic biological samples and lend itself to computer database manipulation, which facilitates DNA comparisons. In 1986, he used the related method of DNA profiling in a confounding case of two brutal rape and murder attacks in Leicestershire, UK. Eventually the police instigated the first DNA-based manhunt, asking for voluntary samples from all men of a certain age in the area’s villages. After a convoluted series of events, which included a false confession, the murderer persuading a colleague to act as a proxy for the blood test, and an overheard conversation in a pub, the police tracked down the killer, who is serving a life sentence for each murder. Forensic teams worldwide now routinely use DNA profiling. It has not only convicted many criminals, but has also absolved innocent people who were wrongly accused. Furthermore, DNA is quite stable and remains relatively intact after death. As a result, scientists could take advantage of the method to name disaster victims, including those of 9/11, and Jeffreys could confirm the identity of an exhumed body thought to be the Nazi war criminal Josef Mengele.
Applications of DNA fingerprinting and related techniques are endless. In bone marrow transplants, for example, the circulating blood cells should carry donor, not recipient, DNA patterns. Scientists can ferret out DNA signatures of inherited diseases and cancers. The method has addressed problems in international smuggling, conservation biology, and molecular anthropology as well. Investigators can establish, for instance, that a wildlife trophy came from the corpse of a protected animal. Furthermore, they can use it to avoid mating close relatives while trying to save an endangered population. Molecular ecologists have harnessed the strategy to figure out which individuals have spawned the most offspring. It has advanced the fields of evolutionary and population biology, enabling detailed genetic comparisons of various groups. For example, Jeffreys and others showed that the breadth of human variation in Africa was considerably greater than that in non-African populations. These observations supported the theory that people originated in Africa.
Southern invented a technology that made complex genomes accessible to meticulous analysis, and Jeffreys capitalized on this method to uncover the huge diversity of genetic variation. The effects of these innovations have been profound — reverberating over a wide range of sociological, medical, scientific, and forensic arenas.
by Evelyn Strauss
Key publications of Alec Jeffreys
Jeffreys, A.J., Wilson V., and Thein, S.L. (1985). Hypervariable “ministatellite” regions in human DNA. Nature. 314, 67–73.
Jeffreys, A.J., Wilson, V., and Thein, S.L. (1985). Individual-specific “fingerprints” of DNA. Nature. 316, 76–79.
Jeffreys, A.J., BrookField, John F.Y., and Semeonoff, R. (1985). Positive identification of an immigration test-case using human DNA fingerprints. Nature. 317, 818–819.
Jeffreys, A.J., Neumann, R., and Wilson, V. (1990). Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell. 60, 473.
Jeffreys, A.J., MacLeod, A., Tamaki, K., Neil, D.L., and Monckton, D.G. (1991). Minisatellite repeat coding as a digital approach to DNA typing. Nature. 354, 204–209.
Jeffreys, A.J. (1993). 1992 William Allan Award Address. Am. J. Hum. Genet. 53, 1–5.
Key publications of Edwin Southern
Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.
Arnhein, N. and Southern, E.M. (1977). Heterogeneity of the ribosomal genes in mice and men. Cell. 11, 363–370.
Southern, E.M. (1982). Application of DNA analysis to mapping the human genome. Cytogenet. Cell Genet. 32, 52–57.
Southern, E.M., Maskos, U., and Elder J.K. (1992). Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics. 13, 1008–1017.
Case-Green, S.C., Mir, K.U., Pritchard, C.E., and Southern, E.M. (1998). Analysing genetic information with DNA arrays. Curr. Opin. Chem. Biol. 2, 404–410.
Southern, E.M. (2000). Blotting at 25. TIBS. 25, 585–588.