This year’s Lasker Award for Basic Medical Research is being shared by two British scientists, Edwin Southern of the University of Oxford and Alec Jeffreys of the University of Leicester, for the “development of two powerful technologies – Southern hybridization and DNA fingerprinting – that together revolutionized human genetics and forensic diagnostics.”
The Basic Medical Research award was one of three 2005 Laskers presented at a luncheon in New York on Sept. 23.
DNA fingerprinting is dependent on having DNA accessible to probes, which is the key accomplishment of southern blotting. Jeffreys developed the technique more or less by accident in the mid-1980s while working on the evolution of the myoglobin gene, which codes for a protein that stores oxygen in muscle once it is released by hemoglobin.
Jeffreys realized that so-called tandem repeat regions of the genome that harbor restriction fragment length polymorphisms, or RFLPs, would be a rich source of individual differences in DNA sequence – much richer than single nucleotide polymorphisms (SNPs). “A SNP can exist in only two states,” he told Diagnostics & Imaging Week. For example, a given SNP might have either an adenine (A) or a thymine (T). This enables three categories, depending on which version is inherited from each parent: AA, TT or AT. For this reason, SNPs are no more useful than blood types for identifying an individual.
In contrast, tandem repeats of DNA have many more possible states, making them useful for identifying individuals. For example, some tandem repeats can have anywhere from 20 to thousands of copies. Jeffreys likened the tandem repeats to beads on a string, with different individuals having different numbers of beads. When the variability across multiple regions is compared simultaneously, the combinatorial possibilities are so great that the chan-ces of unrelated individuals having the same pattern were very small.
With the advent of polymerase chain reaction techniques in the 1990s, DNA fingerprinting was further refined because SNPs could now be identified within the tandem repeats. Now, “not only do people have different numbers of beads, but the beads are different colors,” Jeffreys said. Between the two techniques, each region now has hundred of millions of different possible configurations, making the chance that two people who are not identical twins will show the same DNA fingerprint close to zero.
DNA fingerprinting has multiple applications, such as paternity testing, forensics, and establishing relationships in animal colonies that help optimize breeding programs. Jeffreys says he is proudest of the very first case where his technology was applied practically; in 1985, it was used in a British immigration case to establish conclusively that a child was indeed the son of an immigrant, rather than a nephew, and thus prevented the child’s deportation.
Asked whether he had any worries about the technology, Jeffreys said that its use in forensics, while very helpful both in identifying and clearing suspects, could be overdone. For example, the UK now is storing the DNA samples even of suspects that have been cleared, which, in Jeffreys’ opinion, amounts to “almost a license to the police to go to those ethnic communities who they feel are over-prevalent in criminal behavior” and establish a DNA database.
These and some other applications, he said, need political discussion and oversight; “They have not been discussed by the House of Commons to my knowledge – and I believe they should be.” But overall, he does not have too many concerns, and notes that “every technology has an upside and a downside.”
With the many different applications that DNA fingerprinting made possible, demand for the new technique soon outstripped the possibilities of a university laboratory, Jeffreys said. “At one point, we managed to jam the university switchboard with incoming calls” from people who needed to be typed.
Jeffreys licensed the technology to Imperial Chemical Industries, which in turn formed a subsidiary called Cellmark Diagnostics, with offices in Abingdon, UK and Germantown, Maryland. Cellmark Diagnostics was acquired by Orchid Biosciences (Princeton, New Jersey) in February 2001, and now is known as Orchid Cellmark.
In his Nature Medicine commentary, Jeffreys thanks “all my friends at Cellmark Diagnostics who turned a bit of academic eccentricity into something truly of value” by successfully commercializing the technology. “Without them, large-scale applications would not have arisen,” he said.
DNA fingerprinting is one of many applications that was made possible by southern blotting, a technique for detecting a specific segment of DNA without purifying it from the rest of the genome.
In the 1960s and 1970s, scientists were itching to tackle the mysteries of larger genomes; to this end, a method was necessary to identify specific sequences in DNA fragments.
With smaller genomes, DNA fragments can be separated on an agarose gel; different fragments will migrate at different speeds across this gel according to their size. Large genomes, however, will not separate cleanly because they have too many DNA fragments of similar sizes; instead of nicely separated bands, the DNA forms a more or less continuous blob on the gel.
Theoretically, it is possible to cut out part of one gel (which will contain similar-sized DNA fragments) and further process the fragments so that it is ultimately possible to detect a specific DNA fragment from a very large genome. But it is an extremely time-consuming method.
The key advantage of southern blotting is that it removes the DNA from the gel, thus making it accessible to labels, while preserving the separation by size that the gel has achieved. Essentially, Southern used a filter paper to draw fluid out of the gel, and the DNA fragments moved up along with the fluid; once blotted onto the filter paper, DNA could be labeled with probes.
While southern blotting is named for Southern (though Jeffreys points out in his Nature Medicine commentary that “with typical modesty, Ed never calls them southerns”) the method has spawned two copycat names: northern blotting for RNA and western blotting for a related technique to detect proteins. As of press time, eastern blotting awaited a technology that was worthy of its name. Given the recent interest in epigenetics, perhaps a technology for detecting DNA methylation would be appropriate.