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Improved nonisotopic PCR-SSCP for screening of p53 mutations
Clinical Biochemistry, Vol. 32, No. 3, 233–235, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter
PII S0009-9120(99)00002-8
Improved Nonisotopic PCR-SSCP for Screening of p53 Mutations JUREERUT POOART,1 TEMDUANG LIMPAIBOON,1 and VIRAPHONG LULITANOND2 1
Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand, and 2Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Introduction he p53 tumor suppressor gene encodes a 393 amino acid nuclear phosphoprotein, which acts as a transcriptional activator (1). Mutations of p53 tumor suppressor gene have been described in almost all types of human malignancies including familial cancers (2), therefore, screening for p53 mutations is becoming more and more widely used. In order to avoid labor-intensive sequencing of the complete coding sequence, several pre-screening methods have been developed and applied to the p53 gene. Among them, single strand conformation polymorphism (SSCP) appears to be highly sensitive. SSCP relies on electrophoretic detection of conformation changes in single-stranded DNA molecules resulting from point mutations or nucleotide sequence variations. The method becomes versatile and sensitive with the advent of polymerase chain reaction (PCR). The original SSCP protocol involves the separation of denatured radiolabeled PCR amplimers on large-formatted nondenaturing gels followed by autoradiography (3). However, this method is both time-consuming and cumbersome. In this study, we have improved a rapid, inexpensive and nonisotopic PCR-SSCP method for determining of p53 mutations.
T
ously described (4). The intronic primers specific for exons 5– 8 of the p53 gene were used for amplifying according to Koga et al. (5). Exon 5: 59-CAACTCTGTCTCCTTCCT-39 (forward) and 59-CAGCCCTGTCGTCTCTCCAG-39 (reverse); exon 6: 59-GCCTCTGATTCCTCACTGAT-39 (forward) and 59TTAACCCCTCCTCCCAGAGA-39 (reverse): exon 7: 59-AGGCGCACTGGCCTCATCTT-39 (forward) and 59-TGTGCAGGGTGGCAAGTGGC-39 (reverse); and exon 8: 59-TTCCTTACTGCCTCTTGCTT-39 (forward), and 59-AGGCATAACTGCACCCTTGG-39 (reverse). PCR
CONDITIONS
The final 50 mL PCR mixture contained 1 mL of template DNA, 26 mM of each primer, 10 mM Tris-HCl pH 8.5, 50 mM KCl, 1.5 mM MgCl2 for exons 6 – 8 (2 mM MgCl2 for exon 5), 200 mM each of deoxyribonucleoside triphosphate (dNTP) and 1.25 units of Taq DNA polymerase (Pharmacia Biotech, Uppsala, Sweden). The PCR mixture was heated to 94 °C for 5 min and subjected to 35 cycles of 94 °C for 30 sec, 60 °C for 30 sec and 72 °C for 30 sec. Following cycling, tubes were heated to 72 °C for 7 min. SSCP
Materials and methods DNA
SAMPLES AND PRIMERS
Paraffin-embedded specimens of cervical carcinoma were used as a source of tumor DNA for PCR amplification. Templates were prepared as previCorrespondence: Dr. Temduang Limpaiboon, Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: 66 43 362 028; E-mail: temduang@kku1. kku.ac.th Manuscript received October 16, 1998; revised and accepted December 21, 1998. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
CONDITIONS
Two microliter aliquots of PCR products were added to 10 mL of 90% formamide, 20 mM EDTA, 0.025% bromphenol blue and 0.025% xylene cyanol. Samples were then denatured by heating to 95 °C for 5 min followed by quenching on ice. Ten microliters of each sample were loaded onto a 82 3 80 3 0.75 mm 15% polyacrylamide (69:1 acrylamide to N, N9-methylene-bis-acrylamide); 90 mM Tris-borate (pH 8.0), 2 mM EDTA (TBE) gel. The running buffer for polyacrylamide gel was 1 3 TBE. The electrophoretic separation was carried out on a horizontal electrophoresis apparatus (Mighty Small II SE 250, Hoefer Scientific. Instruments, San Francisco, CA, USA) for 1 1/2 h at 400 V at 15 °C, respectively. 233
POOART, LIMPAIBOON, AND LUILITANOND
Figure 1 — Nonisotopic PCR-SSCP analysis of p53 gene mutations. N, normal DNA; T, tumor DNA. (A) Exon 7. (B) Exon 8. Mutations are indicated by arrowheads.
Figure 2 — Mutations of p53 gene identified on sequencing gel. PCR-amplified DNA of exons 7 and 8 shown in Figure 1 was direct sequenced. (A) Exon 7. (B) Exon 8. Arrowheads indicate positions of mutated bases.
Discussion Single strands were visualized using a silver staining method described by Bassam et al. (6) with slight modifications. Briefly, a gel was fixed for 3 min in 10% ethanol and for 3 min in 1% nitric acid then stained for 10 min in impregnation solution (0.1 g AgNO3, 150 mL formaldehyde in 100 mL deionized water). After staining, the gel was soaked in developing solution (3 g Na2CO3, 150 mL formaldehyde, 100 mL of 2 mg/mL Na2S2O3 in 100 mL deionized water) until the appropriate signal was visualized, then immersed for 5 min in 5% acetic acid to stop the reaction. The gel was then dried and photographed. NUCLEOTIDE
SEQUENCING
Samples showed band shift by SSCP were subsequently sequenced by the dideoxy method (Sequenase® Version 2.0; United States Biochemical, Cleveland, OH, USA). Results Examples of mutations detected in exons 7 and 8 using nonisotopic SSCP are shown in Figure 1. SSCP bands can be clearly discerned in tumor samples, in contrast to only two bands normally seen in normal samples. Direct dideoxy sequencing of tumor PCR products confirmed that single point mutations were present in samples showing polymorphism (Figure 2). Sequencing the products shown in Figure 1A and 1B revealed an ATG 3 TTG transversion of codon 237 (Met 3 Leu) (Figure 2A) and a CGT 3 CCT transversion of codon 273 (Arg 3 Pro) (Figure 2B), respectively. 234
PCR-SSCP analysis has been widely used for the detection of human genetic diseases, somatic mutations of oncogenes and tumor suppressor genes in cancer tissues. However, the conventional isotopic methods still required a great deal of work to perform as routine laboratory tests. Recently, several groups have reported the successful use of nonisotopic SSCP analysis for detection of p53 gene mutations (7–9). In order to improve these previous techniques, we have examined various electrophoretic conditions, such as the gel concentration, the ratio of acrylamide to bisacrylamide, the composition of the buffer of the gel, the voltage, the temperature of the gel, and the presence or the absence of the glycerol in the gel. We found that the best separation was achieved in the shortest time (1 1/2 hours) when the samples were electrophoresed, under the conditions described in this article. The running time of 1 1/2 hours is considerably faster than other reported methods (7–9). Under our analysis conditions, we were able to distinguish between the mutated alleles and the normal one that could be confirmed by direct sequencing. In order to obtain highly sensitive nonisotopic SSCP analysis, we found it is important to optimize PCR conditions to get high yield amplimers and less remaining primers. Our optimized PCR conditions described here offer good PCR products suitable for nonisotopic SSCP analysis. The size of DNA fragment also influences the sensitivity of SSCP, more than 90% of mutations can be detected in DNA fragments up to 200 bp in length (10). In this study each exon was amplified with flanking intron primers and PCR product size of exons 5– 8 were 248, 181, 177, and 231 bp, respectively. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
NONISOTOPIC PCR-SSCP
In conclusion, we have improved the nonisotopic PCR-SSCP method for screening of p53 gene mutations in paraffin-embedded tumor tissues. The technique described here has several advantages: (a) it can be carried out in general laboratories due to no requirement of radioactive materials; (b) the minimal equipment requirements eliminate the problems concerning of handling and cost; and (c) the shortest running time makes this improved SSCP method possible to be performed routinely. Acknowledgements This work was supported by the grants from the National Research Council of Thailand and the Faculty of Medicine, Khon Kaen University.
References 1. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323–31. 2. Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res 1996; 24: 141– 6. 3. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphism of human DNA by gel electrophoresis as single-strand conformation poly-
CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
4.
5.
6. 7.
8.
9.
10.
morphisms. Proc Natl Acad Sci USA 1989; 86: 2766 – 70. Wu AM, Ben-Ezra J, Winberg C, Colombero AM, Rappaport H. Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffin-embedded specimens. Lab Invest 1990; 63: 107– 14. Koga H, Zhang S, Kumanishi T, et al. Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry. Acta Neuropathol (Berl) 1994; 87: 225–32. Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991; 196: 80 –3. Oto M, Miyake S, Yuasa Y. Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus. Anal Biochem 1993; 213: 19 –22. Bosari S, Marchetti A, Buttitta F, et al. Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and non radioactive silver-stained SSCP analysis. Diagn Mol Pathol 1995; 4: 249 –55. Soong R, Iacopetta BJ. A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed paraffin-embedded tumors. Mod Pathol 1997; 10: 252– 8. Hayashi K. PCR-SSCP: a method for detection of mutations. GATA 1992; 9: 73–9.
235
PII S0009-9120(99)00002-8
Improved Nonisotopic PCR-SSCP for Screening of p53 Mutations JUREERUT POOART,1 TEMDUANG LIMPAIBOON,1 and VIRAPHONG LULITANOND2 1
Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand, and 2Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Introduction he p53 tumor suppressor gene encodes a 393 amino acid nuclear phosphoprotein, which acts as a transcriptional activator (1). Mutations of p53 tumor suppressor gene have been described in almost all types of human malignancies including familial cancers (2), therefore, screening for p53 mutations is becoming more and more widely used. In order to avoid labor-intensive sequencing of the complete coding sequence, several pre-screening methods have been developed and applied to the p53 gene. Among them, single strand conformation polymorphism (SSCP) appears to be highly sensitive. SSCP relies on electrophoretic detection of conformation changes in single-stranded DNA molecules resulting from point mutations or nucleotide sequence variations. The method becomes versatile and sensitive with the advent of polymerase chain reaction (PCR). The original SSCP protocol involves the separation of denatured radiolabeled PCR amplimers on large-formatted nondenaturing gels followed by autoradiography (3). However, this method is both time-consuming and cumbersome. In this study, we have improved a rapid, inexpensive and nonisotopic PCR-SSCP method for determining of p53 mutations.
T
ously described (4). The intronic primers specific for exons 5– 8 of the p53 gene were used for amplifying according to Koga et al. (5). Exon 5: 59-CAACTCTGTCTCCTTCCT-39 (forward) and 59-CAGCCCTGTCGTCTCTCCAG-39 (reverse); exon 6: 59-GCCTCTGATTCCTCACTGAT-39 (forward) and 59TTAACCCCTCCTCCCAGAGA-39 (reverse): exon 7: 59-AGGCGCACTGGCCTCATCTT-39 (forward) and 59-TGTGCAGGGTGGCAAGTGGC-39 (reverse); and exon 8: 59-TTCCTTACTGCCTCTTGCTT-39 (forward), and 59-AGGCATAACTGCACCCTTGG-39 (reverse). PCR
CONDITIONS
The final 50 mL PCR mixture contained 1 mL of template DNA, 26 mM of each primer, 10 mM Tris-HCl pH 8.5, 50 mM KCl, 1.5 mM MgCl2 for exons 6 – 8 (2 mM MgCl2 for exon 5), 200 mM each of deoxyribonucleoside triphosphate (dNTP) and 1.25 units of Taq DNA polymerase (Pharmacia Biotech, Uppsala, Sweden). The PCR mixture was heated to 94 °C for 5 min and subjected to 35 cycles of 94 °C for 30 sec, 60 °C for 30 sec and 72 °C for 30 sec. Following cycling, tubes were heated to 72 °C for 7 min. SSCP
Materials and methods DNA
SAMPLES AND PRIMERS
Paraffin-embedded specimens of cervical carcinoma were used as a source of tumor DNA for PCR amplification. Templates were prepared as previCorrespondence: Dr. Temduang Limpaiboon, Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: 66 43 362 028; E-mail: temduang@kku1. kku.ac.th Manuscript received October 16, 1998; revised and accepted December 21, 1998. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
CONDITIONS
Two microliter aliquots of PCR products were added to 10 mL of 90% formamide, 20 mM EDTA, 0.025% bromphenol blue and 0.025% xylene cyanol. Samples were then denatured by heating to 95 °C for 5 min followed by quenching on ice. Ten microliters of each sample were loaded onto a 82 3 80 3 0.75 mm 15% polyacrylamide (69:1 acrylamide to N, N9-methylene-bis-acrylamide); 90 mM Tris-borate (pH 8.0), 2 mM EDTA (TBE) gel. The running buffer for polyacrylamide gel was 1 3 TBE. The electrophoretic separation was carried out on a horizontal electrophoresis apparatus (Mighty Small II SE 250, Hoefer Scientific. Instruments, San Francisco, CA, USA) for 1 1/2 h at 400 V at 15 °C, respectively. 233
POOART, LIMPAIBOON, AND LUILITANOND
Figure 1 — Nonisotopic PCR-SSCP analysis of p53 gene mutations. N, normal DNA; T, tumor DNA. (A) Exon 7. (B) Exon 8. Mutations are indicated by arrowheads.
Figure 2 — Mutations of p53 gene identified on sequencing gel. PCR-amplified DNA of exons 7 and 8 shown in Figure 1 was direct sequenced. (A) Exon 7. (B) Exon 8. Arrowheads indicate positions of mutated bases.
Discussion Single strands were visualized using a silver staining method described by Bassam et al. (6) with slight modifications. Briefly, a gel was fixed for 3 min in 10% ethanol and for 3 min in 1% nitric acid then stained for 10 min in impregnation solution (0.1 g AgNO3, 150 mL formaldehyde in 100 mL deionized water). After staining, the gel was soaked in developing solution (3 g Na2CO3, 150 mL formaldehyde, 100 mL of 2 mg/mL Na2S2O3 in 100 mL deionized water) until the appropriate signal was visualized, then immersed for 5 min in 5% acetic acid to stop the reaction. The gel was then dried and photographed. NUCLEOTIDE
SEQUENCING
Samples showed band shift by SSCP were subsequently sequenced by the dideoxy method (Sequenase® Version 2.0; United States Biochemical, Cleveland, OH, USA). Results Examples of mutations detected in exons 7 and 8 using nonisotopic SSCP are shown in Figure 1. SSCP bands can be clearly discerned in tumor samples, in contrast to only two bands normally seen in normal samples. Direct dideoxy sequencing of tumor PCR products confirmed that single point mutations were present in samples showing polymorphism (Figure 2). Sequencing the products shown in Figure 1A and 1B revealed an ATG 3 TTG transversion of codon 237 (Met 3 Leu) (Figure 2A) and a CGT 3 CCT transversion of codon 273 (Arg 3 Pro) (Figure 2B), respectively. 234
PCR-SSCP analysis has been widely used for the detection of human genetic diseases, somatic mutations of oncogenes and tumor suppressor genes in cancer tissues. However, the conventional isotopic methods still required a great deal of work to perform as routine laboratory tests. Recently, several groups have reported the successful use of nonisotopic SSCP analysis for detection of p53 gene mutations (7–9). In order to improve these previous techniques, we have examined various electrophoretic conditions, such as the gel concentration, the ratio of acrylamide to bisacrylamide, the composition of the buffer of the gel, the voltage, the temperature of the gel, and the presence or the absence of the glycerol in the gel. We found that the best separation was achieved in the shortest time (1 1/2 hours) when the samples were electrophoresed, under the conditions described in this article. The running time of 1 1/2 hours is considerably faster than other reported methods (7–9). Under our analysis conditions, we were able to distinguish between the mutated alleles and the normal one that could be confirmed by direct sequencing. In order to obtain highly sensitive nonisotopic SSCP analysis, we found it is important to optimize PCR conditions to get high yield amplimers and less remaining primers. Our optimized PCR conditions described here offer good PCR products suitable for nonisotopic SSCP analysis. The size of DNA fragment also influences the sensitivity of SSCP, more than 90% of mutations can be detected in DNA fragments up to 200 bp in length (10). In this study each exon was amplified with flanking intron primers and PCR product size of exons 5– 8 were 248, 181, 177, and 231 bp, respectively. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
NONISOTOPIC PCR-SSCP
In conclusion, we have improved the nonisotopic PCR-SSCP method for screening of p53 gene mutations in paraffin-embedded tumor tissues. The technique described here has several advantages: (a) it can be carried out in general laboratories due to no requirement of radioactive materials; (b) the minimal equipment requirements eliminate the problems concerning of handling and cost; and (c) the shortest running time makes this improved SSCP method possible to be performed routinely. Acknowledgements This work was supported by the grants from the National Research Council of Thailand and the Faculty of Medicine, Khon Kaen University.
References 1. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323–31. 2. Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res 1996; 24: 141– 6. 3. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphism of human DNA by gel electrophoresis as single-strand conformation poly-
CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999
4.
5.
6. 7.
8.
9.
10.
morphisms. Proc Natl Acad Sci USA 1989; 86: 2766 – 70. Wu AM, Ben-Ezra J, Winberg C, Colombero AM, Rappaport H. Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffin-embedded specimens. Lab Invest 1990; 63: 107– 14. Koga H, Zhang S, Kumanishi T, et al. Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry. Acta Neuropathol (Berl) 1994; 87: 225–32. Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991; 196: 80 –3. Oto M, Miyake S, Yuasa Y. Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus. Anal Biochem 1993; 213: 19 –22. Bosari S, Marchetti A, Buttitta F, et al. Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and non radioactive silver-stained SSCP analysis. Diagn Mol Pathol 1995; 4: 249 –55. Soong R, Iacopetta BJ. A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed paraffin-embedded tumors. Mod Pathol 1997; 10: 252– 8. Hayashi K. PCR-SSCP: a method for detection of mutations. GATA 1992; 9: 73–9.
235