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Clinical Chemistry 43: 2251-2255, 1997;
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(Clinical Chemistry. 1997;43:2251-2255.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Single-strand conformation polymorphism analysis to detect p53 mutations: characterization and development of controls

Judith A. Welsh1, Katariina Castrén2 and Kirsi H. Vähäkangas2,a

1 Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, MD 20892 (address for reprint requests: fax 301-496-0497; e-mail welshj{at}intra.nci.nih.gov)

2 Department of Pharmacology and Toxicology, University of Oulu, FIN-90220 Oulu, Finland.
a Author for correspondence. Fax 358-8-537-5247; e-mail kirsi. vahakangas{at}oulu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified p53 exons 4-8, where mutations frequently occur in human tumors. Easily obtainable and clearly distinguishable positive controls were developed by replacing the regular 5' primers in amplification with primers that include one to three mutated sites. Careful purification of the amplified products by gel electrophoresis appeared to be essential. The efficiency of the method was studied by using previously sequenced samples with p53 mutations and the various positive controls. The use of two temperatures (exon 4: 4 °C and 15 °C; other exons: 4 °C and 20 °C) in combination with other optimized conditions resulted in 98% efficiency in mutation detection, which was considered sufficient for routine screening.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
p53 is the most frequently mutated gene in human cancers, and >7000 mutations in various cancer types have been published (1)(2)(3)(4). The spectrum of p53 mutations varies considerably between different tumor types and different etiologies. This suggests that p53 is an important contributing factor in the development of many human cancers. Increasingly, data are emerging that p53 mutations are clinically important (2) and there are strong implications, especially in breast cancer, that mutational status and mutational position in the p53 gene have prognostic value (5)(6).

Well-controlled, reproducible methods to prescreen for p53 mutations are greatly needed in clinical oncology to avoid extensive sequencing of wild-type samples. Single-strand conformation polymorphism (SSCP) analysis of amplified p53 exons appears to be useful and is already being used widely for this purpose. Newly emerging sequencing techniques may, however, simplify mutational screening by sequencing in the future (5). So far, SSCP analysis has also been reported to be more sensitive in detecting mutations than sequencing (7)(8)(9). While many of the p53 studies still utilize the original radioactive SSCP method (10), silver-staining for detection of p53 mutations is being used in increasing frequency (11)(12)(13)(14). Optimization of the SSCP conditions is essential for analytical sensitivity and efficiency (15)(16)(17)(18). Although several hundred papers have been published with SSCP analysis to detect p53 mutations, we could not find one in which the authors had made a systematic effort to optimize all the critical points: conditions with several temperatures, primers to detect full exons with splice sites, and positive controls for all strands amplified. The requirement for confirmation of any found mutation by an independently amplified DNA should be self-evident because of the possibility of polymerase errors (18)(19).

Here we describe a complete set of conditions and the development of positive controls for a temperature-controlled nonradioactive SSCP analysis to examine mutations within exons 4–8 of the p53 tumor-suppressor gene. Mutations in the p53 gene occur not only in the coding region but also at the intron–exon splice sites (2). In contrast to some other published primer sets (14), our primers enable characterization of the splice sites as well as the exon sequences, making this method complete for all the changes in and around p53 exons 4–8.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Two sets of intronic primers, the second set internal to (nested with) the first one for each exon, were used for amplification ((20), Table 1 ). Either Dynazyme (Finnzymes) or AmpliTaq (Perkin-Elmer) was used in conditions described earlier (19). Amplified PCR products were gel-purified on a gel with 40 g/L NuSieve 3:1 agarose (FMC Bioproducts). DNA was eluted in 0.5 mL of 0.5 mol/L ammonium acetate, precipitated with ethanol, dried, and resuspended in H2O. For SSCP analysis, the samples were mixed with Stop solution (US Biochemicals) and heat-denatured. The PhastSystem®, PhastGel Homogeneous 20% polyacrylamide gels, PhastGel Native Buffer Strips, and PhastGel DNA Silver Staining Kit were purchased from Pharmacia Biotech. Gels were preserved with a Gel Drying Kit from Promega.


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  		Table 1. List of the internal (nested) intronic primers used in this study.

The electrophoretic conditions were optimized for exon 7 by varying temperature (2, 4, 10, 15, and 20 °C), voltage (200, 300, and 400 V), and running time. Criteria for optimized conditions were the separation and visibility of bands and the detectability of mutations. For exon 7, 20 °C and 4 °C gave clearly better band patterns than the other temperatures, and the use of both temperatures increased the efficiency greatly over the use of either temperature alone. Optimal electrophoresis conditions for exon 7 were 450 average volt hours (aVh) at 20 °C and 550 aVh at 4 °C at 200 V. Except for voltage (200 V), the electrophoresis conditions for the other exons varied, depending on the size of the fragment (exon 5: 500 aVh at 20 °C and 600 aVh at 4 °C; exon 6: 300 aVh at 20 °C and 400 aVh at 4 °C; exon 8: 500 aVh at 20 °C and 600 aVh at 4 °C), but otherwise the same conditions as for exon 7 were applicable for exons 5, 6, and 8. The prerun conditions (400 V, 10 mA, 1 W, and 100 aVh) and the conditions for sample application (25 V, 10 mA, 1 W, and 2 aVh) suggested by the manufacturer were better than any other of the combinations tested and were used for all five p53 exons.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Exon 4 contains several polymorphic sites, of which codon 72 is the most common (21) and has been implicated as a risk factor for lung cancer (22)(23)(24). To differentiate the codon 72 polymorphism from exon 4 mutations, SSCP conditions were optimized with the use of patient's samples [Castrén et al., submitted for publication] and BEAS 13 cell line (a derivative of BEAS 2B, which contains both codon 47 and codon 72 polymorphisms) (25). The results were confirmed by digestion with BstUI (New England Biolabs), which does not digest a polymorphic sample. The best running conditions were 15 °C with 700 aVh and 4 °C with 800 aVh. A codon 72 polymorphism could be detected at both 20 °C and 15 °C. Interestingly, all positive controls for exon 4 showed band shifts at 15 °C but not at 20 °C, which was a further justification for the use of 15 °C. Both BEAS 13 cell line with polymorphism (lane 8 in Fig. 1 a) and one of the mutations were detectable only at 4 °C or 8 °C, thus proving the need for the lower temperature in the analysis of exon 4. Because 4 °C was used for the other exons, it was selected for routine use. Usually the codon 72 polymorphism is detected with restriction fragment analysis (22)(23)(24). In restriction analysis in general it is difficult to get a completely digested sample, which creates the relative difficulty in differentiating homozygous nonpolymorphic samples with incompletely digested fragments from heterozygous samples. Such difficulties are avoided in SSCP analysis, where fragments from both alleles run separately (Fig. 1a ). Similar results on the codon 72 polymorphism have been obtained by others with nonradioactive SSCP (12)(26).



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  		Figure 1. SSCP analysis of p53 exons.

dsC, double-strand control; ssC, single-strand control. The positive controls with artificially introduced mutations are listed in Table 1Up . a, Polymorphisms and artificially mutated controls created for SSCP analysis for exon 4. Lane 1, ssC; lane 2, X4A1; lane 3, X4A2; lane 4, X4A3; lane 5, ssC; lane 6, wild-type patient sample with heterozygous codon 72; lane 7, wild-type patient sample with homozygous codon 72 polymorphism; lane 8, BEAS 13 cell line containing both homozygous codon 72 polymorphism and codon 47 polymorphism. b, Comparison of artificially mutated controls created for SSCP analysis of p53 exon 5. Lanes 1 and 7, ssC; lane 2, X5A1; lane 3, X5A2; lane 4, X5A3; lane 5, X5A4; lane 6, X5A5; lane 8, X5A5. c, Exon 8. Lane 1, dsC; lanes 2, 3, 6, and 7, mutated samples; lanes 4 and 8, ssC; lane 5, X8A1.

The only way to judge whether the assay is successful is to use positive controls in each assay. Soto and Sukumar (27) used different breast cancer cell lines with p53 mutations as positive controls for their SSCP method. A different strategy was adopted in this study enabling the use of any wild-type DNA as the template in DNA amplification. A systematic effort was taken to develop easily obtainable and clearly distinguishable positive controls by designing several artificially mutated primers for each exon with one to three nucleotide changes at various positions in the 5' primer (Table 1Up ). Lymphocyte DNA isolated from a healthy individual was amplified by these and the normal right-side primers (3' on the coding strand). In these amplified products the nucleotide change in the primer provided the mutation, while the rest of the sequence remained normal. Enough positive controls were developed until easily detectable controls were found for both electrophoresis conditions of each exon (Table 1Up ). Not all of these designed positive controls gave mobility shifts in both of the studied temperatures. In fact, for some exons it was quite laborious to find suitable primers for both temperatures (Table 1Up ). Especially for exons 5 and 6, all positive controls were detectable at 4 °C (Fig. 1bUp ), whereas many remained undetectable at 20 °C (Table 1Up ). As negative controls, p53 exons were amplified from lymphocyte DNA with the use of normal primers and confirmed wild-type by sequencing.

Generally, the band patterns of the same samples were very similar among gels, even when done in two different laboratories, but at times slight variations occurred in different gels, emphasizing the need of controls in each gel. We also noticed that impurities in amplification reactions would show as additional bands in SSCP, thus complicating the interpretation of the results. Careful gel purification of the amplified product was essential to avoid this problem.

The theoretical basis of SSCP is far from clear. The conformation of a denatured single-stranded fragment or its mobility on a nondenaturing gel cannot be predicted in advance (15). Theoretically, the simplest band pattern includes 5 bands if both mutated and wild-type sequences are present—one band per each single-stranded DNA sequence and one band for the double-stranded DNA, which in some systems may appear between single-stranded bands. In our study, the double-stranded bands run off the gel. Because the conformation changes according to the temperature, more than one conformation may occur close to the critical transformation temperature, and consequently, more single-stranded bands may be seen (10)(18)(28). Similarly, some mutations are detectable only at a very narrow temperature limit. Careful optimization and the need for analyzing DNA samples under several conditions in SSCP is acknowledged in the literature (15)(18)(29)(30). Taking this into consideration, many of the radioactive methods have used two to three different sets of conditions (10)(13)(26)(31). The automated temperature control in the PhastSystem is a major advantage compared with room or cold-room temperatures, which can never be strictly regulated.

In our study, one temperature was not sufficient to detect all the different mutations at hand (Fig. 1Up ). For instance, in exon 8 one of the artificially mutated controls (lane 5, Fig. 1cUp ) and one of the mutations (lane 7, Fig. 1cUp ) could be differentiated from the wild-type (lane 4, Fig. 1cUp ) only at 20 °C. Two temperatures in combination with other optimized conditions resulted in an efficiency of 98% and thus were considered sufficient for a routine screening method. Only 1 of the 43 p53 exon sequences with known mutations could not be detected, whereas all of the artificially mutated positive controls were detectable at least at one temperature (Table 1Up ). If only one temperature had been used, 16% (7 of 43) of the mutations (or 19%; 12 of 63, if we include the positive controls in the calculations) would have been missed at higher temperatures (15 °C for exon 4 and 20 °C for other exons), and 28% (12 of 43) at 4 °C (21%; 13 of 63, with the positive controls). Further increasing the number of electrophoresis running conditions, however, would not have given significant advantage, considering the increased amount of work for a small increase in the efficiency.

There was no effect of the site of a mutation on the overall efficiency if only the known exon mutations were considered. However, at higher temperatures it was more difficult to detect artificially mutated samples, where the mutations are located near the end of the exon. Two different mutations within the codon 280 in exon 8 obviously caused very different conformation changes: AGA to AAA was detectable at 20 °C, but AGA to AGG was not detectable at either 20 °C or 4 °C.

In conclusion, we achieved high efficiency for p53 mutation detection by our SSCP by careful development of optimal conditions, which include two temperatures for each exon. This method is now being used in both our experimental (32) and clinical studies. The designed positive controls guarantee the reproducibility of this method, which is essential for the clinical work.


   Acknowledgments
 
We thank Virna de Benedetti, Brenda Gerwin, Marc Greenblatt, Ylermi Soini, and Mohammed Aslam Khan for providing sequenced p53 mutated samples. We are indebted to Brenda Gerwin, Kathy Forrester, and Curtis C. Harris for the critical reading of the manuscript at various stages and to Helen Cawley for giving us the protocol for the use of the gel-drying kit. Financial support from the Finnish Cancer Societies (K.H.V.) and a special grant for biotechnology from the University of Oulu (K.H.V.) are acknowledged. The Ida Montin Foundation, Urho Kankanen Foundation, the Finnish Cancer Societies, and the Biomedical Graduate School at the University of Oulu have provided travel grants for K.C.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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