3.2.1. Introduction to SSCP
For identification o f known mutations, such simple tests as restriction endonuclease digestion or detection by ASOs that are described above can easily be applied to large numbers of individuals. In some instances previously unidentified sequence changes may be found, as is described in Section 3.1.3. above, where new mutations were identified as a new restriction pattern oïDdéV digests and as heteroduplexes. These are coincidental findings, but on a broader scale a systematic approach is needed to identify the underlying, currently unknown, sequence changes. Major gene rearrangements are still identified by Southern blotting but in the case of point mutations, other methods have to be used. The four techniques mainly used are; chemical cleavage of mismatch (CCM), denaturing gradient gel electrophoresis (DGGE); direct sequencing; or single strand conformation polymorphism (SSCP).
These published methods all allow comparison o f the sequence o f specific fragments o f DNA amplified in vitro by PGR (Saiki et al 1985) from different individuals. The first uses chemical cleavage o f mismatched bases (CCM) in the duplex formed between two heterologous DNA fi*agments after hybridisation (Montandon et al 1989). This is a robust technique which has been used successfully to look for mutations in the apoB gene (Dunning et al 1991). Because it is based on chemical methods, the technique is able to detect all mismatched bases irrespective of sequence (Michaud et al 1992), and individuals can be identified who are heterozygous for any sequence difference compared to the normal "probe" DNA, which is radiolabelled with [a-
^^]dCTP. Fragments of DNA of about 500-600 bp give good results, but for longer fragments, the yield o f amplified DNA is reduced, and cleaved mismatches within 50-60 bp o f the ends of the DNA fragments may not be detected (as a size reduction). This can be overcome by using amplifying oligonucleotides that produce fragments that overlap by 100 bp. The method involves several time-consuming steps, some involving hazardous chemicals and is thus not feasible for large number o f samples.
A second approach is the use of denaturing gradient gel electrophoresis (Myers et al 1985a), which again appears to be able to detect all possible mutations (Cariello and Skopek 1993). DGGE depends upon the difference in melting profile conferred by a single base substitution. DGGE employs a linear gradient o f urea/formamide in a polyacrylamide gel where the DNA migrates into an increasing concentration of dénaturant (Cariello and Skopek 1993). It is possible to predict the behaviour of a DNA fragment in a denaturing gradient gel, solely from the basepair sequence (Lerman & Silverstein 1987), and compute a calculated melting map. DGGE is only sensitive to mutations in the low-temperature melting domain o f a molecule. The melting characteristics o f a molecule can be altered by adding GC rich sequences to it, the so called GC clamp (Myers et al 1985b and 1985c). The method works best if a GC-rich sequence clamp is introduced at one end o f the sequence (e.g.included on one PCR primer). As for chemical cleavage, the technique is somewhat inconvenient to set up and use. A recent report has used this method to screen the promoter region of the LDL receptor for mutations in 500 FH patients, but none were identified (Top et al 1992). In another study, Lombardi et al (1993) examined 32 patients with FH and used DGGE to detect a variation in 27 o f the patients.
difficult to use on large genes in large number o f samples without a pointer as to where in the gene the mutation is to be found. The quality of the sequencing needs to be very high as a common problem is observing terminations in more than one track, which results in the detection o f "heterozygotes" caused by artefacts. Of the two methods used, the Sanger chain termination method (Sanger et al 1977), that relies on the ability o f dideoxynucleotides, which lack the 3'- hydroxyl group critical to phosphoribosyl chain extension, to terminate a strand synthesis from a specific oligonucleotide priming site by a DNA polymerase, is much more popular and easier than the Gilbert Maxam method, which uses chemical cleavage at specific bases (Maxam and Gilbert 1977). Automated DNA sequencers perform electrophoresis and detection on gels similar to manual sequencing gels, but the detection system is usually fluorescence, either using oligonucleotides with fluorescent labels attached, or incorporating fluorescent nucleotides into the product during PCR. With automated sequencing machines, this is a method preferred by some for a primary search for unknown mutations.
SSCP is an increasingly popular method to use for detection o f unknown mutations, mainly because it is relatively simple to use and has the potential for mutation detection in a large number o f samples. It has been an established technique for many years to separate the two strands o f a duplex by non-denaturing electrophoresis (Maniatis et al 1982). However, the idea and demonstration that non-denaturing electrophoresis would resolve single nucleotide polymorphisms in either of the two strands of a duplex is much more recent (Orita et al 1989a). Under non-denaturing conditions a single strand o f DNA will adopt a conformation (presumably dependent on internal base-pairing between short segments by foldback) which is uniquely dependent on its sequence composition. This conformation will usually be different if even a single base is changed, although the theoretical basis for the folding is not well understood.
SSCP is a method capable of identifying a large proportion o f sequence variations in a single strand o f DNA, typically between 150 and 250 nucleotides in length (Hayashi 1991, Sheffield et al 1993). Most conformations seem to alter the physical configuration or size enough to be detectable as a mobility difference upon electrophoresis though a retarding matrix such as acrylamide gel. The ability to detect differences in the migration pattern is reduced in fi'agments over 300-400 bp.
In many laboratories [a-^^P]dCTP is incorporated during the PCR, and diluted denatured PCR product is electrophoresed on a "long" (40 cm) acrylamide gel, but a number o f systems using shorter gels and non-radioactive methods for detection have been developed (Mohaber et al 1991, Hongyo et al 1993), such as fluorescence (Makino et al 1992) and silver staining (Ainsworth et al 1991). Thus, SSCP is to date the easiest method to use for identification of unknown mutations in a large number of samples.
3.2.2. Methods: Adaption of SSCP for the 3* part of exon 4
SSCP was performed as described in Chapter 2, Section 2.4.4. A wide variety o f different conditions for SSCP were evaluated, including track length, temperature and the presence or absence o f glycerol in the gel. The specific conditions are described in the results below.
The samples used to carry out the SSCP evaluation for the 3' part o f exon 4 were the AGI 97, D206E, Fs206 and C210X.
3.2.3. Results
3.2 3.1. The effect of track length
Figure 3.2.1., 3.2.2., and 3.2.3. show the increased resolution o f th e single strands w ith increased gel tra c k length.
M SS HD OS Origin --- Origin 516 bp ___ _ 506 bp --- 394 bp --- ^ 344 bp --- 200 bp --- --- 10
Figure 3.2.1. SSCP analysis on a small gel format; 6 cm long, 0.8 mm thick and 7% polyacrylamide, 10% glycerol, 1:49 bisacrylamide:polyacrylamide. The DNA is detected by silver staining. The double stranded DNA fragment is 234 bp long. SS and DS denote single strands and double strands, respectively and the relative positions are shown by the transverse lines. The relevant sizes o f the 1 kb ladder (M) are displayed on the left o f the Figure, and the sizes o f the double stranded DNA is presented on the right. The samples in lanes I - 4 and 6 are all boiled. The first sample is the 2 bp deletion (Fs206), the second is the 3 bp deletion (AG 197), 3 is the C210X, 4 is the D206E, lane 6 contains a control sample. In lane 5 the 1 kb ladder is used as a marker. Lanes 7-10 show the native 2 bp, 3 bp and the D206E and control, in that order.
The smallest gel (Figure 3.2.1.), the double stranded D N A samples have run 4.3 cm after 5 hours o f electrophoresis at 10 mA and room tem perature, the single stranded D N A has run 3.2 cm and 3.5 cm. Lanes 1-4 and 6 show sam ples denatured by boiling in form am ide and SD S. B o th the deletions show heteroduplexes (lanes 1,2 and 7,8) as does the D 2 0 6 E m utation (lanes 4 and 9). There is no indication o f a difference in the m igration o f the single strands, except for the low er
strand o f the 2 bp deletion (lane 1). The upper heteroduplex band for the 3 bp deletion falls on the slower migrating single strand for that sample and for that sample tw o bands can be seen o f the double strand reflecting the 3 bp difference in size.
M SS