APLICACIÓN TEMPORAL DE LOS BENEFICIOS PENITENCIARIOS

Large rearrangements can be conveniently detected by anafysing total cell DNA directly, using Southem blotting techniques or by m ultiplex PCR methods. Southem blotting (Southem, 1975) involves the fragmentation of genomic DNA by digestion w ith restriction enzymes and separation by agarose gel electrophoresis. The DNA is transferred from the gel to a nylon membrane by Southem blotting and hybridised to a radiolabelled DNA probe to detect size alterations. It has been used to detect mutations in many disorders, including Hunter disease (Froissart et oL, 1993). This method does not require specific sequence information for analysis and detects the presence of all large rearrangements. Point mutations can be detected if they alter a restriction site (Bemstein et oL, 1989). Disadvantages are that it is not always Informative for small rearrangements and it requires a large amount of undegraded DNA sample.

M ultiplex PCR reactions amplify a complete gene sequence, in several large fragments, using genomic DNA as a template. Size alterations are then detected by electrophoretic separation through an agarose gel, (e.g. in Fabiy disease, Komreich

et oL, 1993). Sequence information is required for analysis but it allows detection without requiring a gene-specific probe and w ith m inimal template DNA. As w ith Southem blotting, small rearrangements might not be detected.

1.3.7.1.3 Detection of small sequence alterations by alterations in the DNA conformation

PCR am plification of specific areas of genomic DNA or reverse-transcribed RNA (cDNA), followed by analysis of their conformation, which is sequence dependent, is the basis of several m utation detection methods. In each of the following methods, sequence changes cause alterations in the electrophoretic migration of DNA, which are detected on an acrylamide-based gel system using autoradiography or non­ isotopic visualisation. The wild-type DNA is anatysed in an adjacent lane to the m utant sequence.

(a) Double-stranded conformation analysis, DSCA, (Saad et oL, 1994) detects altered migration of homoduplex DNA by electrophoresis though a non-denaturing pofyacrylamide gel.

Cb) In heteroduplex analysis (Keen et aL, 1991; W hite et oL, 1992) the DNA for analysis is melted and annealed w ith normal DNA so that the m utant alleles form incompletely paired DNA duplexes. These are separated by electrophoresis through a non-denaturing aciylamide gel and their migration is retarded relative to the homoduplexes.

(c) In single-strand conformation polymorphism, SSCP (Orita et a l, 1989; Hayashi, 1991) analysis, the DNA is denatured and alterations in secondcuy structure are detected by electrophoresis in a non-denaturing polyacrylamide gel.

A ll three methods require m inim al DNA template. Heteroduplex analysis and SSCP analysis detect small rearrangements and point mutations w ith more than 80% sensitivity in DNA that is less than BOObp in length, using optimised conditions. The gel systems are simple and the DNA does not require modification, except by dénaturation for SSCP detection and for the formation of heteroduplexes. The m ain disadvantage is that some sequence alterations are not detected.

1.3.7.1.4 Detection of small sequence changes by other methods

In denaturing gradient gel electrophoresis, DGGE, (reviewed by Cariello et aL,

1993; Fodde et aL, 1994) DNA is amplified by the PCR and is anatysed by migration th ro u ^ a potyacrylamide gel that contains a linearty increasing gradient of dénaturant, such as urea and formamide. The homoduplex DNA is progressively denatured un til it reaches a concentration which is equivalent to the m elting temperature of the lowest m elting domain. At this point the migration is severely retarded. Since sequence variations in the highest m elting temperature domains are often not resolved, a G /C ta il is sometimes incorporated into the PCR product to create a high m elting temperature domain, ensuring that mutations are in a lower m elting domain. The sensitivity of the method is enhanced by mixing m utant and normal DNA to form heteroduplexes, in addition to homoduplex DNA (Sheffield

et aL, 1989). The major disadvantage is that the preparation of the denaturing gel requires more expertise than for anatysis of changes in the DNA conformation.

The RNase cleavage method (Myers et aL, 1985) uses a radio-labelled wild-type, RNA probe which is hybridised to the sample DNA, forming an RNA-DNA heteroduplex. Areas of mismatch are digested w ith RNase A. which cleaves single­

stranded RNA. and these are separated by electrophoresis on an agarose gel. The requirement for radioactively labelled RNA and its low detection efficiency, about 50%, have lim ited its use.

In chemical mismatch cleavage (Cotton et aL, 1988; Smooker et aL, 1993), DNA is amplified by PCR. The radio-labelled wild-type DNA is mixed w ith the either unlabelled or labelled sample (Forrest et aL, 1991) to form heteroduplexes and base changes are detected by cleavage of mismatches. DNA analysis is by electrophoretic separation through a polyacrylamide gel. Hydroxylamine and osmium tetroxide bind to unpaired cytosine and thymine bases, respectively, and the DNA can then be cleaved w ith piperidine. The method detects close to 100% of m utations, although T-G mismatches, which are the most stable, are not always successfully cleaved. Alternative mismatch-detection methods use a carbodiimide (CDI) to modify guanine and thymine bases. The DNA surrounding the base modified w ith CDI can be cleaved w ith E. coli ABC excinuclease for electrophoretic separation. DNA-bound CDI can also be detected by binding to antibodies. Alternatively and more usually, mis-matched bases that bind CDI can be detected by PCR am plification from a primer towards the modified base, in a 5’ to 3’ direction. The pofymerase carmot extend beyond the modified base, thus creating a shortened PCR-product. The m utation can be localised, an advantage of this method over the methods exploiting changes in DNA conformation. Disadvantages are the toxicity of the chemical reagents and the requirement for mismatch modification steps prior to electrophoretic analysis.

1.3.7.1.5 Mutation screening strategy used in this study

SSCP analysis was chosen as the method for detection of unknown mutations in the a-galactosidase A gene. Although it is less efficient than chemical mismatch detection and does not allow localisation of the m utation w ithin the sequence analysed, the sim plicity of SSCP analysis is a distinct advantage. Its sensitivity is higher than RNase cleavage and equivalent to heteroduplex analysis. DGGE is complicated by the use of a gel w ith a gradient of dénaturant and requires expertise regarding the analysis of sequence melting domains and the use of (X)-clam ps. Genomic DNA was used as the template since most of the samples for anafysis were in this form rather than RNA. As there are onfy 7 exons in the gene, PCR am plification of the protein-coding region in seven reactions is not a problem.