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Technical Briefs |
a author for correspondence: fax 33-1-53-72-50-48; e-mail helene.blanche{at}cephb.fr
PCR–single-strand conformation polymorphism (SSCP) analysis is an attractive technique used to screen for unknown mutations because of its simplicity and widespread applicability (1). The technique relies on single-nucleotide variations modifying the conformation of single-stranded DNA and therefore its mobility in polyacrylamide gels. The detection of these conformers is performed either by autoradiography or by nonisotopic methods such as silver-staining (2), ethidium bromide-staining (3), chemiluminescence (4), or fluorescence (5).
In this paper, we report a nonisotopic PCR-SSCP method with the use of the Pharmacia MultiPhorTM (Pharmacia Biotech) electrophoresis unit for sensitive, reproducible, and cost-effective experiments that can be performed at high throughput. This method was established to analyze the 12 exons of the glucokinase (GCK) gene to identify mutations involved in maturity-onset diabetes of the young, a subset of non-insulin-dependent diabetes.
The 12 GCK exons (6) were amplified by PCR (fragments ranging from 145 to 367 bp), as reported previously (2), either on GeneampTM 9600 (Perkin-Elmer) or PTC100TM (MJ Research) DNA thermal cyclers.
Forty-six known mutations (6)(7)(8) were used to establish
the optimal electrophoretic conditions for the 12 PCR fragments. Two
types of precast polyacrylamide gels, allowing the analysis of 34 and
23 DNA samples, respectively, were used: nondenaturing gels
[Cleangel-HP®, 10% total acrylamide (T) concentration,
2% total extent of cross-linking (C); ETC Elektrophorese-Technik] and
partially denaturing gels (Excelgel®, 7.5% T, 3% C,
Pharmacia Biotech). Electrophoresis was performed at temperatures of
6–20 °C, the length of assays depending on size and GC content of
the PCR fragments. For all exons except exon 9, samples were run on
Cleangel-HP, rehydrated in Delect® (ETC
Elektrophorese-Technik) gel buffer, with electrode wicks soaked in
Delect anode and cathode buffers. Electrophoresis on Cleangel-HP was
carried out in three steps: prerun (200 V, 20 mA, 10 W) and run (375 V,
30 mA, 20 W), followed by a last step to refine bands (450 V, 30 mA, 20
W). For exon 9, Excelgel and sodium dodecyl sulfate buffer {200
mmol/L Tricine [N-tris(hydroxymethyl)methylglycine], 200
mmol/L Tris, 5.5 g/L sodium dodecyl sulfate} were used, and a
one-step run was performed: 550 V, 30 mA, 20 W. Specific
electrophoretic conditions (assay time and temperature) were determined
for each exon (Table 1
). Gels were stained with a silver-staining kit (Silver Staining
Kit Plus One®; Pharmacia Biotech) and then wrapped in
cellophane (soaked in a solution of 100 mL/L glycerin and 100 mL/L
acetic acid) for preservation.
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All nucleotide changes previously identified (8) were
detected. Fig. 1
shows the electrophoretic profile of exon 10. The effect of
different electrophoretic conditions was evaluated from the number of
bands characteristic of a given PCR product, their sharpness, and
resolution. The purification of our PCR products tested on exons 6, 9,
and 10 (data not shown) did not provide an increase in sensitivity,
contrary to other published results (9). Indeed with
specific PCR products, in addition to two conformers, bands resulting
from the interactions between PCR primers and the single-stranded DNA
were visualized and provided an easier analysis of the electrophoretic
profile.
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The protocol used for the GCK gene allowed us to establish a successful
strategy for the development of PCR-SSCP on other genes such as
BRCA1 (breast cancer 1, data not shown) on the basis of GC
content and the length of PCR products. We found high sensitivity,
especially in fragments generally difficult to test with the use of the
PCR-SSCP technique, i.e., larger than 250 bp and presenting a high GC
content (
60%). Lower detection limits and reliability of the
detection of mutations by this PCR-SSCP analysis may be explained by
multiple factors: (a) the use of precast gels;
(b) the efficient temperature regulation with an independent
thermostatic circulator that provides a wide range of precise running
temperatures from 6 to 20 °C and avoids the addition of glycerol to
the gels often reported as decreasing the effects of temperature
variability (10); and (c) the use of partially
denaturing conditions with a temperature varying from 6 to 12 °C,
which increases the sensitivity of PCR-SSCP analysis, particularly for
large or GC-rich fragments (exon 9). We hypothesize that mild
denaturing conditions extend the exposed surface area of
single-stranded DNA, which tends to assume a folded configuration in
the complete absence of denaturing agents. Therefore, detection of
locally confined structural differences in PCR fragments is improved.
Thus, the lack of detection often encountered with the PCR-SSCP analysis, as compared with other current technologies for the study of large fragments, is overcome in these conditions. Moreover, screening for mutations on the MultiPhor system is not expensive considering the gel-loading capacity, apparatus, and reagent prices as compared with fluorescent methods that require an automatic sequencer. Contrary to isotopic PCR-SSCP analysis, which is less reproducible, only a single run is routinely required to screen each exon.
In conclusion, PCR-SSCP analysis on the MultiPhor appears to be a useful and reliable tool in screening for unknown sequence variations at a high throughput and is especially adapted for laboratories that cannot perform fluorescent PCR-SSCP analysis on an automatic sequencer. An easy, nonradioactive detection method is required more than ever because of genetic diagnosis programs, in which routine screening for mutations will become more important for healthcare purposes.
Acknowledgments
We are grateful to M. F. Legrand for technical assistance and to clinicians and patients who send us blood samples. We thank Howard Cann for helpful discussions about the manuscript. This study was funded by the "Ministère de L'Education nationale, de L'Enseignement supérieur et de la Recherche" and the "Conseil Régional d'Ile de France."
Footnotes
Fondation Jean Dausset-CEPH, 27 rue Juliette Dodu, 75010 Paris, France
References
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C. *, additional
conformers present on samples carrying a mutation.