Detection of known mutations

4.3.1. Restriction analysis

The simplest way of detecting a mutation is by restriction enzyme digestion if it creates or destroys a restriction site. Restriction analysis consists of a short incubation of PCR amplified DNA with a restriction endonuclease, followed by electrophoresis on either an agarose or a polyacrylamide gel matrix. Restriction sites may also be introduced to or abolished from a specific target DNA sequence by using either or both sense and antisense mismatched PCR primers (Saiki et al. 1985; ODell et al. 1996). Throughput may be increased considerably by the use of MADGE technology (Day and Humphries,

1994).

4.3.2. Allele specific oligonucleotides (ASO)

When the allelic sequence variation is known, it is possible to synthesise short oligonucleotides, that are complementary to wild-type or mutant allele. An ASO will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridisation under appropriate conditions. (Saiki et al. 1986). DNA is dot- blotted on a membrane for subsequent hybridisation with the two complementary AS Os. A higher throughput approach to this method has been developed for the detection of multiple mutations (Shuber et al. 1993); this involves multiplex PCR of the appropriate exons, followed by dot-blot hybridisation with pooled ASOs.

4.3.3. Amplification refractory mutation system (ARMS)

The basis of this system is that oligonucleotides which are complementary to a given DNA sequence, except for a mismatch at the 3’ hydroxyl residue, will not function as primers in a PCR reaction under appropriate conditions (Newton et al. 1989). Typically the test consists of two separate but complementary reactions; the first reaction contains an ARMS primer specific for the normal DNA sequence, and the second, a primer

specific for the mutant sequence. A normal homozygote generates a PCR product from only the normal primer, the heterozygote from both normal and mutant ARMS primer, and a mutant homozygote from only the mutant primer. This system therefore allows genotyping solely by inspection of PCR products by gel electrophoresis.

4.3.4. Profile o f oligonucleotide dissociation gel electrophoresis (PODGE)

In principle, this is similar to ASO, but in the PODGE approach, electrophoresis is the means of separating bound from free oligonucleotide and the temperature is the wash stringency variable (Day et al. 1995b). An oligonucleotide is annealed to a DNA target sequence and its melting profile is determined as the temperature rises over a set range. An oligonucleotide that is a perfect match will remain bound for the longest time and will therefore dissociate last, while an oligonucleotide bound to a mutant heterozygote or homozygote will be less stable due to the mismatch and will dissociate first. Thus in a heterozygote, three bands are obtained in order of decreasing migration: (1) never bound oligonucleotide (migrating as a free species for the duration of the run), (2) oligonucleotide released at a lower temperature because it has a mismatch with the mutant PCR strand to which it is attached and (3) oligonucleotide release at the highest temperature because it was annealed with perfectly cognate strands in the PCR product. The greater the difference between the normal and mutant strands, the earlier the release of the mismatched oligonucleotide with respect to the perfectly bound oligonucleotide, and consequently the greater the migration distance between the dissociated oligonucleotide spots. The advantages of this method include visualisation of full melting profile of oligonucleotide and no predetermination of the melting temperature is necessary. There is no need for prior knowledge of the mutant sequence and potentially it is a high throughput method, each track is an ‘all-in-one’ result (thus it is possible to see mutant and normal spots in one track). Multiplexing is also feasible. This method does however, in its present set up, require autoradiographic detection, and has not been used for de novo mutation detection.

4.3.5. The ‘one best m ethod’ and future improvements.

An ideal mutation detection system will be miniaturised, provide high throughput and be easily integrated in parallel processes, enabling many analyses to be performed simultaneously. In summary, certain features must be present in the ideal mutation

DNA, a 100% detection rate, no false positive or negative results, non-complex equipment, a single step approach involving no harmful chemicals, no electrophoresis, high throughput and low cost. The method should not be time consuming and should detect all unknown mutations. The method of use should also depend on the need and experience of the user e.g. those needing 100% detection such as in a diagnostic set up and those tolerant of 80% detection such as in the research laboratory. Other points for consideration are a need to avoid radioactivity, occasional versus constant need for mutation detection and the application to which mutation detection is being used e.g. diagnostic, candidate gene or spectrum of disease mutations.

Systems for mutation detection must therefore be automated, miniaturised whenever possible, accurate, low-cost, user friendly and have high throughput. Improvements in the areas of sample preparation and acquisition, assay technology, detection systems and data management and analysis need to be made. In the areas of sample preparation and assay, miniaturisation is necessary, sample sizes must be reduced by a factor of ten at least. This will require substantial automation of the processes used. Currently electrophoresis is used in most methodologies, but methods relying on DNA hybridisation and morphology are also being employed. Along with miniaturisation, a reduction in PCR amplification time is necessary. Using conventional sample wells and cycling apparatus, each cycle takes about two minutes. This can be reduced by an order of magnitude as can the total volume being amplified. With miniaturisation, an improvement in detection steps is required such as the use of confocal microscopy and mass spectrophotometry. Better systems will need to be made available to handle this increased information rapidly and efficiently.