State of the art in 1989

Less than 20 years ago (20 years is the potential maximum patent term, with some exceptions), the state of genome research and nucleotide sequence-based diagnostics was relatively new. While the first discovery of a molecular, completely hereditary trait, the ABO blood group, occurred in the early 1900s, subsequent discovery and cataloguing of additional polymorphisms 1 in humans was slow and tedious, limited primarily by the analytical techniques. Until the mid-to-late 1970s essentially all polymorphisms were identified using biological reagents (e.g. antibodies) or electrophoresis. The tediousness and difficulties inherent in these techniques is reflected in the detection of only about 250 polymorphic proteins up until the end of the 1970s.

The explosion of new molecular technologies that began in the mid-1970s had a profound effect on identifying polymorphisms. Two techniques in particular markedly advanced the ability to identify polymorphisms: restriction enzyme digestion and DNA sequencing technologies. With the increasing commercial availability of restriction enzymes and the successful application of whole genome Southerns for more complex organisms, restriction fragment length polymorphisms (RFLPs) became a popular and preferred way to detect differences between individuals. Anyone who has tried their hand at these techniques can attest however to the difficulties and frustrations in obtaining clean and consistent results. Luckily, in the late 1970s, DNA sequencing was pioneered, which escalated the number of polymorphisms catalogued. With the further advent of polymerase chain reaction (PCR) in 1985, the information explosion magnified.

Using these techniques, different types of polymorphisms were characterized. Single base changes in coding regions of genomic DNA, especially those that resulted in amino acid changes, have long been sought after as a source of polymorphism, especially for diagnostic testing. Restriction enzyme mapping, and especially DNA sequencing of genomic DNA, allowed analysis of non-coding DNA regions as well, expanding the available pool of polymorphisms and facilitating the discovery of new types of polymorphisms, such as variable lengths of repeat DNA. For diagnostic testing however, the main reliance remained on detecting nucleotide differences in the coding sequence of the genes themselves. These changes that result in a different amino acid basis can be the basis for various diseases and syndromes. For example, a single amino acid change in each chromosomal copy of beta-haemoglobin results in sickle cell anaemia. So to test for carriers of one chromosomal copy, because the nucleotide change was not detectable as a restriction site polymorphism, it was necessary to determine the actual sequence of the individual’s beta-haemoglobin genes or to perform hybridization studies with oligonucleotides, a technique that is notoriously difficult to detect single base differences. The advent of polymerase chain reaction (PCR) vastly improved the ease and reliability of assaying single nucleotide polymorphisms. Oftentimes, however, testing of family members was still required for a conclusive diagnosis.

With the exception of rare restriction fragment length polymorphisms in linkage disequilibrium with some important disease-associated alleles 2, non-coding DNA was not generally regarded as useful for detecting polymorphic coding alleles. Apparently only one group found value in finding and using non-coding region polymorphisms to indicate the haplotype of a gene family.3