Single strand conformation analysis (SSCA) is a technique developed for the
detection of single base changes in DNA. It was first published in 1989 (Orita et al., 1989a) using total human genomic DNA which was digested using frequent cutting
restriction enzymes, denatured, electrophoresed under non-denatuiing conditions, blotted
on to a membrane, hybridised with a radioactively labelled probe DNA and detected using
autoradiography. On the resulting autoradiograph two bands were observed due to the
coding and non-coding strands. Band shifts were seen in samples with single base
differences, probably due to different confomiations taken up by the single stranded
DNA. The relative mobility of the single strand confoiTners could conveniently be altered
by changing the percentage of glycerol in the gel and / or the temperature at which the gel
was electrophoresed.
Development of the polymerase chain reaction (PCR) (Saiki eta l., 1985) which allows the specific amplification of a sequence defined by two flanking oligonucleotide
primers led to the simplification of this technique. The use of a labelled substrate in the
PCR reaction allowed direct autoradiography of the dried gel removing the need for
restriction enzyme digestion, blotting and hybridisation (Orita et al., 1989b). The size of the fragment analysed could now be controlled by experimental design, which is
advantageous as sequences less than 400 base pairs long have been shown to be more
informative (Hayashi, 1991). When this PCR based technique was applied to point
mutations previously described in N-ras and K-ras (Orita et a l, 1989b), it was found to detect each of the mutations in both products analysed (the length of the PCR products
analysed were 103 and 162 base pairs in length for N-ras and K-ras respectively). SSCA
analysis of known Alu repeat elements showed 17 out of 43 to be polymorphic
(39.5%)(Orita et at., 1990), while similar analysis of 16 anonymous Alu repeats found 9 to be informative (56.3%)(lizuka et a l, 1992).
The dependence of the detection of a given variation on use of different
electrophoretic conditions has been investigated by various workers. These factors were
reviewed by Hayashi (1991). Although this paper recommended the use of low cross
linker concentrations (49:1, aci-ylamide: bis-aciylamide), 29:1 and 19:1 have successfully
been used by a number of workers (for example Michaud et al., 1992). Adding glycerol to the gel mix has been found to aid detection of variation in most cases, but not all. For
example in the ornithine 5-aminotransferase gene del C, G > A and T > C were only
detected without glycerol in the gel mix whereas two different G > A changes, G > C and
C > T were not visible on gels without glycerol (Michaud et al., 1992). Use of a concentration of 5% glycerol in the gel mix, rather than 10% as in the original protocol, was shown to allow detection of more variation in the human papilloma virus genome
(Spinai'di et a i, 1991). Temperature has also been shown to have a dramatic effect on the migration of the different single strand conformers and hence on the bands seen.
Room temperature or 4"C have been the most commonly used temperatures published but
some workers suggest an an angement of fans to keep the temperature of the
electrophoresis down (A-L Boiresen, personal communication) or water jacketing to
maintain constant temperature (marketed by Stratagene). Halving the ionic strength of the
buffer has also been shown to have an effect on the bands seen and their relative mobility
(Spinardi et al., 1991).
More recently Hayashi and Yandell (1993) have reviewed the sensitivity and
methods used for SSCA and report that the sensitivity can approach 100% under
optimised electrophoresis conditions using fragments up to 300 base pairs in length.
The effect of sequence context as well as gel composition on the sensitivity of SSCA has
been studied using variations in the mouse beta-globin gene. M ost base changes were
detected in the purine rich strand and the optimum fragment length range was found to be
120-2(K) base pairs (Glavac & Dean, 1993). A selection of variations detected by SSCA
A simple technique called heteroduplex analysis has been suggested to be com
plementary to the SSCA approach in the detection of variation (White et al., 1992). The PCR product is denatured and slowly let cool, allowing the strands to re-anneal. If a
single base change is present, a certain amount of the product will re-anneal to the other
allele. This heteroduplex contains a single base mismatch, which on electrophoresis has
a slower mobility than either of the homoduplex DNAs formed when the strands re
anneal to their 'conect ' partner. Although this technique was performed separately from
SSCA analysis, it was suggested that both techniques could be attempted using the same
radioactively labelled sample (White et a i, 1992). Furthermore, various workers have shown that heteroduplexes produced when two DNA samples which differ by as little as
a single base change are mixed can be detected by ethidium bromide staining (for example
Wilkin et al., 1993) or silver staining (for example Friedl et al., 1993).
Non-radioactive SSCA has been published by a few groups, either using
ethidium bromide detection (for example Hongyo et a i, 1993) or using silver staining (Tsai et al., 1993). Interestingly, one group has shown, by silver staining, an aberrant double stranded band in addition to an altered SSCA pattern in individuals with a AG
deletion (Zhong et al., 1993). Non-radioactive SSCA has also been published where use was made of the Phast-System (Phanuacia) using pre-made gels (for example Mohabeer
et al., 1991; March et al., 1993b). This has been suggested to be more suitable for use in the clinical laboratory setting as the reproducibility of the results should be higher.
Another modification of this technique which was claimed to increase sensitivity
was published independently by two groups. The modification involves transcribing the
region of interest as RNA, which is done by incoiporating phage promoter sequences
into the PCR primers. Analysis is then perfomied in a similar gel system to that
noiTnally used for DNA-SSCA. One of the groups reported a 50% increase in the
amount of variation detected (Sarkar et al., 1992), whereas the otlier reported two cases where a variation was detected that had not been detected using DNA-SSCA (Danenberg
gene associated condition base changes detected reference
CFTR cystic fibrosis G > A X 4 Plieth eta l..
(non deletion A508 type) G > T x 2 C > T x 1 T > G X 1 G > C x 1 A > C X 1
1992
N F l neurofibrom atosi s del 2 ins 1
Zhong et a l, 1993
Factor IX haemophilia B C > T Demers et
a l , 1990 PKU phenylketonuria C > T x 2 T > G x 1 G > A X 2 del 3 Labrune et a l , l 9 9 l
OAT hereditary deficiency of G > A X 6 Michaud et
ornithine Ô- T > C x 4 a l , 1992 aminoiransferasc C > A X 1 A > G X 2 G > T x 3 G > C x 4 C > T X 5 G > A X 1
single base insertion x 1 deletions x 3 del 3 bps X 1 CHM Choroiderema CC > G X 1 C > A X 1 G > T x 1 del 1 X 1 del 4 bp X 1 Hurk et a l, 1992 Figure 1.3.2.1
E xam ples o f genes where SSCA has been su ccessfully used to iden tify mutations / variations. In each case ihe number of each type of base change detected is identified.