TY - JOUR
AU - Bellanné-Chantelot,, Christine
AB - 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. 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. 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. Fondation Jean Dausset-CEPH, 27 rue Juliette Dodu, 75010 Paris, France Table 1. Electrophoretic conditions for PCR-SSCP analysis on the MultiPhor. Exon . Size, bp . GC content, % . Temperature, °C . Assay time . 1a 195 57.0 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 1b 149 57.0 20 Step 1 15 min Step 2 1 h Step 3 5 min 1c 145 54.5 20 Step 1 15 min Step 2 1 h Step 3 5 min 2 290 62.4 15 Step 1 20 min Step 2 3 h Step 3 15 min 3 295 61.7 9 Step 1 30 min Step 2 2 h Step 3 20 min 4 272 59.9 9 Step 1 30 min Step 2 2 h Step 3 20 min 5 195 64.1 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 6 176 57.4 20 Step 1 15 min Step 2 1 h 15 min Step 3 10 min 7 285 65.9 15 Step 1 20 min Step 2 2 h 45 min Step 3 15 min 8 263 63.4 9 Step 1 30 min Step 2 2 h Step 3 20 min 91 367 67.0 12 4 h 10 263 66.3 20 Step 1 15 min Step 2 1 h 20 min Step 3 15 min Exon . Size, bp . GC content, % . Temperature, °C . Assay time . 1a 195 57.0 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 1b 149 57.0 20 Step 1 15 min Step 2 1 h Step 3 5 min 1c 145 54.5 20 Step 1 15 min Step 2 1 h Step 3 5 min 2 290 62.4 15 Step 1 20 min Step 2 3 h Step 3 15 min 3 295 61.7 9 Step 1 30 min Step 2 2 h Step 3 20 min 4 272 59.9 9 Step 1 30 min Step 2 2 h Step 3 20 min 5 195 64.1 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 6 176 57.4 20 Step 1 15 min Step 2 1 h 15 min Step 3 10 min 7 285 65.9 15 Step 1 20 min Step 2 2 h 45 min Step 3 15 min 8 263 63.4 9 Step 1 30 min Step 2 2 h Step 3 20 min 91 367 67.0 12 4 h 10 263 66.3 20 Step 1 15 min Step 2 1 h 20 min Step 3 15 min 1 Run on Excelgel 7.5%; all others analyzed on Cleangel-HP 10%. Table 1. Electrophoretic conditions for PCR-SSCP analysis on the MultiPhor. Exon . Size, bp . GC content, % . Temperature, °C . Assay time . 1a 195 57.0 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 1b 149 57.0 20 Step 1 15 min Step 2 1 h Step 3 5 min 1c 145 54.5 20 Step 1 15 min Step 2 1 h Step 3 5 min 2 290 62.4 15 Step 1 20 min Step 2 3 h Step 3 15 min 3 295 61.7 9 Step 1 30 min Step 2 2 h Step 3 20 min 4 272 59.9 9 Step 1 30 min Step 2 2 h Step 3 20 min 5 195 64.1 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 6 176 57.4 20 Step 1 15 min Step 2 1 h 15 min Step 3 10 min 7 285 65.9 15 Step 1 20 min Step 2 2 h 45 min Step 3 15 min 8 263 63.4 9 Step 1 30 min Step 2 2 h Step 3 20 min 91 367 67.0 12 4 h 10 263 66.3 20 Step 1 15 min Step 2 1 h 20 min Step 3 15 min Exon . Size, bp . GC content, % . Temperature, °C . Assay time . 1a 195 57.0 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 1b 149 57.0 20 Step 1 15 min Step 2 1 h Step 3 5 min 1c 145 54.5 20 Step 1 15 min Step 2 1 h Step 3 5 min 2 290 62.4 15 Step 1 20 min Step 2 3 h Step 3 15 min 3 295 61.7 9 Step 1 30 min Step 2 2 h Step 3 20 min 4 272 59.9 9 Step 1 30 min Step 2 2 h Step 3 20 min 5 195 64.1 20 Step 1 15 min Step 2 1 h 10 min Step 3 5 min 6 176 57.4 20 Step 1 15 min Step 2 1 h 15 min Step 3 10 min 7 285 65.9 15 Step 1 20 min Step 2 2 h 45 min Step 3 15 min 8 263 63.4 9 Step 1 30 min Step 2 2 h Step 3 20 min 91 367 67.0 12 4 h 10 263 66.3 20 Step 1 15 min Step 2 1 h 20 min Step 3 15 min 1 Run on Excelgel 7.5%; all others analyzed on Cleangel-HP 10%. Figure 1. Open in new tabDownload slide PCR-SSCP analysis of a GC-rich DNA fragment of the GCK gene. SSCP profiles on exon 10: Lane 1 corresponds to a negative control, lanes 2–4 show samples carrying, respectively, mutations Stop466Leu, Ala450Thr, and Ser498–1G→C. *, additional conformers present on samples carrying a mutation. Figure 1. Open in new tabDownload slide PCR-SSCP analysis of a GC-rich DNA fragment of the GCK gene. SSCP profiles on exon 10: Lane 1 corresponds to a negative control, lanes 2–4 show samples carrying, respectively, mutations Stop466Leu, Ala450Thr, and Ser498–1G→C. *, additional conformers present on samples carrying a mutation. 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.” 1 Hayashi K. PCR-SSCP. 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Crossref Search ADS PubMed 10 Teschauer W, Mussack T, Braun A, Waldner H, Fink E. Conditions for single strand conformation polymorphism (SSCP) analysis with broad applicability: a study on the effects of acrylamide, buffer and glycerol concentrations in SSCP analysis of exons of the p53 gene. Eur J Clin Chem Clin Biochem 1996 ; 34 : 125 -131. PubMed © 1997 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Optimization of Nonisotopic PCR–Single-Strand Conformation Polymorphism Analysis
JF - Clinical Chemistry
DO - 10.1093/clinchem/43.11.2190
DA - 1997-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/optimization-of-nonisotopic-pcr-single-strand-conformation-BFUQCkTtDM
SP - 2190
EP - 2192
VL - 43
IS - 11
DP - DeepDyve
ER -