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Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector
Clinica Chimica Acta 301 (2000) 205–211 www.elsevier.com / locate / clinchim
Short communication
Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector a, b c d Zhenzhong Zhang *, Yiming Wu , Weiming Cheng , Rujin Wu a
Department of Pharmacy, Henan Medical University, Zhengzhou, 450052, China College of Public Health, Henan Medical University, Zhengzhou, 450052, China c Thrombus Hospital of Taikang, Taikang, 471400, China d China Pharmaceutical University, Nanjing, 210009, China
b
Received 1 December 1999; received in revised form 8 May 2000; accepted 15 May 2000
Abstract Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis–single strand conformation polymorphism (CE–SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE–SSCP is rapid, automated, and has high performance. 2000 Elsevier Science B.V. All rights reserved. Keywords: K-ras gene; Mutation; SSCP; Capillary electrophoresis; PCR
1. Introduction Lung cancer is one of the most prevalent cancers and the leading cause of cancer death in the world. The incidence of lung cancer is increasing dramatical*Corresponding author. Tel.: 186-371-6973-994. E-mail address: [email protected] (Z. Zhang). 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00310-7
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Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
ly in recent years. In order to find early biomarkers for lung cancer, it is necessary to study the mechanism of disease progression. Mutations in cellular ras gene have been strongly implicated in various stages of mammalian tumorigenesis. In human tumors, the point mutations which have been identified have largely been localized to codon 12,13 or 61 of three ras genes [1,2]: H-ras, K-ras and N-ras. Traditionally, mutations of these genes were detected by hybridization with radio-labelled DNA probes, allele-specific PCR and restriction fragment length polymorphism with polyacrylamide gel electrophoresis (PAGE). Many studies showed that gene mutations (such as base substitution, point mutation, deletion and insertion) could lead to a conformational change of single-stranded DNA, so the mobility shifts could be observed on the electrophoretogram [3,4]. However, the conventional method is laborious, timeconsuming and has low sensitivity. Capillary electrophoresis (CE) is rapid, automated, has high performance and reproducibility, and has recently been used to detect gene mutations. Single strand conformational polymorphism (SSCP) [5–7], restriction fragment length polymorphism (RFLP) [8,9], liagase chain reaction (LCR) [10], dideoxy fingerprinting amplification refractory mutation system (DFARM) [11], and chemical mismatch cleavage (CMC) [12] have been used to analyze gene mutations by CE. In the present work, SSCP was used with CE and laserinduced fluorescent (LIF) detector to detect the mutation of K-ras gene.
2. Materials and methods
2.1. Apparatus A P/ACE capillary electrophoresis instrument with laser-induced fluorescent detector ( lex 5 488 nm, lem 5 513 nm) was used. A fused silica capillary (37 cm 3 75 mm) was precoated by use of the method of Hjerten [13] with modifications.
2.2. Materials Acrylamide, N, N, N9, N9-tetramethylenediamine (TEMED) and ammonium peroxydisulfate (APS) were purchased from Sigma Chemical Co. 2-Amino-2(hydroxymethyl)-1,3-propanediol (Tris), boric acid, PCR Markers, Taq DNA polymerase, dimethyl sulfoxide (DMSO), blue phenol, xylene cyanole FF and formamide deionized were obtained from Sino-America Biotechnology Co. (SABC). dNTP was from Promega. Thiazole Orange (Fluorescent Molecular Probe) was supplied by Beckman. Primers were synthesized by CyberSyn Biotechnology Corporation (USA).
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
207
2.3. Preparation of sieving medium The short chain linear polyacrylamide gel (SLPA) was synthesized in our laboratory. The molecular mass and viscosity coefficient of SLPA were 2.563 10 5 and 1.25310 23 m 3 .kg 21 respectively. The different concentrations of SLPA were obtained by dissolving SLPA in running buffer (containing 100 mmol / l Tris 1100 mmol / l boric acid 12 mmol / l EDTA15% glycerol12 mg / l thiazole orange), using a magnetic stir bar and stir plate to stir the solution until SLPA was dissolved completely.
2.4. Amplification of K-ras gene by PCR DNA was isolated from lung tissues of rats, which included two groups (normal and tumor), by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook [14] with minor modifications. Primers used in the study were as the following: upstream primer: 59-ACA / TGT / TCT /AAT / ATA / GTC /AC-39, downstream primer: 59-AAA / GAA / TGG / TCC / TGC / ACC /AG-39. The PCR reactions were performed in a 50 ml buffer containing 50 pmol of each primer, 100 pmol of dATP, dCTP, dGTP, dTTP, 5 ml of 103PCR buffer, 0.5 mg template and 8% DMSO. Prior to PCR, the mixture was predenatured at 958C for 5 min and then 1.5 U DNA polymerase was added to the above solution. All PCR reactions were performed in a DNA cycle of SABC (Sino-America Biotechnology Company) and the reaction mixture was covered by 100 ml wax. The reaction mixture was subject to 30 cycles at 948C for 60 s, 648C for 50 s and 728C for 53 s, the extension time for the last cycle was 5 min at 728C, the length of DNA fragment generated by PCR was 212 bp.
2.5. SSCP by non-denatured PAGE Taking 10 ml of PCR products into equal volume of sample buffer containing 20 mmol / l EDTA, 96% formamide deionized, 0.05% bromphenol blue and 0.05% xylene cyanole FF, the above solution was heated at 978C for 5 min and then immediately chilled in ice water bath for 5 min; 10 ml of the above solution was applied to each lane of non-denatured polyacrylamide gel, which consisted of 5% polyacrylamide and 0.4% PEG. Electrophoresis was performed at 200 V for 4 h at 48C and the gel was stained with silver.
2.6. SSCP by capillary electrophoresis The inner surface of the capillary was coated by use of the method of Hjerten [13] with some modifications. Four percent short-chain linear polyacrylamide gel containing 100 mmol / l Tris plus 100 mmol / l boric acid, 2 mmol / l EDTA,
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Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
5% glycerol and 2 mg / l thiazole orange was filled into a capillary coated by a commercial capillary electrophoresis instrument (Beckman) and preconditioned in running buffer consisting of 100 mmol / l Tris, 100 mmol / l boric acid, 2 mmol / l EDTA, 5% glycerol and 2 mg / l thiazole orange for 10 min. After each run the capillary was purged with water for 5 min and again filled with 4% short linear chain polyacrylamide. Prior to electrophoresis, the sample was diluted 6-fold with sample buffer (20 mmol / l EDTA and 96% formamide deionized) and heated at 958C for 7 min and then immediately put into ice water bath for 5 min. After mixing of 0.1 ml thiazole orange with the above sample solution, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under the constant voltage of 8 kv. The DNA fragments were detected by LIF detector ( lex 5488 nm, lem 5513 nm) and the data were processed by Gold System Software.
3. Results In the study, the PAGE–SSCP results showed no mobility shift in the electrophoretogram for the 20 normal cases. A mobility shift did appear for samples 10 and 12 in the tumor group (16 cases). The migration pattern of K-ras gene from normal and tumor lung tissues is shown in Fig. 1. There were
Fig. 1. Electrophoretogram of SSCP for K-ras gene. N: Normal; M: Mutation. Tumor: lane 1–9; Control: lane 10.
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
209
Fig. 2. Electrophoretogram of SSCP by CE for K-ras gene. Upper: tumor (sample 10); Bottom: normal. N: normal; M: mutation.
abnormal band in the lane 1 (sample 10) and lane 3 (sample 12) in the electrophoretogram. This result demonstrated that K-ras gene of sample 10 and 12 mutated in the extron 1. The PCR products of normal and tumor sample were subject to SSCP by CE. The base resolution of two single strands of DNA fragments was achieved with short chain linear polyacrylamide at the concentration of 4%, samples 10 and 12 had 3 peaks in the electrophoretogram between 16.5 min and 18.5 min, and the result showed that samples 10 and 12 had one abnormal peak compared with the normal sample (Figs. 2, 3). Both results of PAGE–SSCP and CE–SSCP were identical.
4. Discussion Several water-soluble polymers, including cellulose derivatives, non-crosslinked linear polyacrylamide and poly (ethylene oxide) (PEO), have successfully been applied to the separation of dsDNA by CE [15]. However, cellulose derivatives and PEO were unsuitable for SSCP analysis due to inadequate resolution or high viscosity at the concentration required for adequate resolution. Short chain linear polyacrylamide has the advantage over long chain linear polyacrylamide due to its low viscosity and adequate resolution and could be filled into the capillary by use of a commercial capillary electrophoresis instrument.
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Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
Fig. 3. Electrophoretogram of SSCP by CE for K-ras gene. Upper: normal; Bottom: tumor (sample 12). N: normal; M: mutation.
Prior to SSCP analysis, the samples were heated to melt the double-stranded DNA molecules into a single strand. Because of the complementary nucleotide sequence of the two single strands of one DNA molecule, their folded conformation was different and this obviously led to different mobility in the non-denatured polyacrylamide gel. The amplification of K-ras gene by PCR generated a DNA fragment of 212 bp in the extron 1. Activation of K-ras oncogene is related to the pathogenesis of various human tumors. The incidence of ras gene mutation in the lung cancer is about 20%. Point mutations which lead to ras activation were confined to the codons 12, 13 and 61 of the gene. Point mutations in codon 12 are commonly seen. Both results of PAGE–SSCP and CE–SSCP demonstrated that K-ras gene of samples 10 and 12 mutated in the extron 1. One abnormal peak was observed respectively in electrophoretogram of PAGE and CE, so this result showed that only one type of mutation appeared for samples 10 and 12. Comparing CE–SSCP with PAGE–SSCP, CE–SSCP has higher resolution than PAGE–SSCE and the former method has the advantages of simplicity, rapidity, automation, high sensitivity and good suitability for clinical analysis.
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
211
References [1] Burmer GC, Rabinovitch PS, Loeb LA. Analysis of c-Ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA sequencing. Cancer Res 1989;49:2141–6. [2] DeFeo-Jones D, Scolnick EM, Koller R, Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature 1983;306:707–9. [3] Larsen LA, Andersen PS, Kanters JK, Jacobsen JR, Vuust J, Christiansen M. A single strand conformation polymorphism / heteroduplex method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome. Clin Chim Acta 1999;280:113–5. [4] Russeva MG, Janakiev PJ, Kirov SA et al. A simple method for detection of factor V R506Q (Leiden) mutation in dried blood spots. Clin Chim Acta 1999;284:89–92. [5] Kiyonori K, Keiko K, Wataru C, Sadao I, Moritoshi K. Fluorescence-based polymerase chain reaction–single-stranded conformation polymorphism analysis of p53 gene by capillary electrophoresis. J Chromatogr A 1996;744:311–20. [6] Jurgen G, Tanja W, Marion B, Markus N, Kurt O, Wolfgang H. Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis. J Chromatogr B 1999;724:239–47. [7] Arakawa H, Nakashiro S, Maedo M, Tsuji A. Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis. J Chromatogr A 1996;722:359–68. [8] Koay ES, Zhu M, Wehr T et al. Detection of apolipoprotein E genotype by capillary electrophoresis. Talanta 1998;45:673–81. [9] Gelfi C, Righetti P, Brancolini V, Cremonesi L, Ferrari M. Capillary electrophoresis in polymer networks for analysis of PCR products: detection of DF508 mutation in cystic fibrosis. Clin Chem 1994;40:1603–5. [10] Jing C, Mann AS, Keith RM, Larry JK, Peter W. Analysis of ligase chain reaction products amplified in a silicon glass chip using capillary electrophoresis. J Chromatogr A 1996;732:151–8. [11] Arakawa H, Vetanala K, Maeda M. Analysis of PCR by capillary electrophoresis and its application to the detection of single base substitution in genes. J Chromatogr A 1994;664:89–94. [12] Ren J, Ulvik A, Refsum H, Ueland PM. Chemical mismatch cleavage combined with capillary electrophoresis: detection of mutations in exon 8 of the cystathionine b-synthase gene. Clin Chem 1998;44:2108–14. [13] Hjerten S. High performance electrophoresis: elimination of electroendosmosis and solute adsorption. J Chromatogr 1985;347:191–8. [14] Sambrook J, Fitch EF, Manitatis T. In: A laboratory manual, New York: Cold Spring Harbor Laboratory Press, 1990, p. 467. [15] Righetti PG, Gelfi C. Non-isocratic capillary electrophoresis for detection of DNA point mutations. J Chromatogr B 1997;697:195–205.
Short communication
Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector a, b c d Zhenzhong Zhang *, Yiming Wu , Weiming Cheng , Rujin Wu a
Department of Pharmacy, Henan Medical University, Zhengzhou, 450052, China College of Public Health, Henan Medical University, Zhengzhou, 450052, China c Thrombus Hospital of Taikang, Taikang, 471400, China d China Pharmaceutical University, Nanjing, 210009, China
b
Received 1 December 1999; received in revised form 8 May 2000; accepted 15 May 2000
Abstract Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis–single strand conformation polymorphism (CE–SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE–SSCP is rapid, automated, and has high performance. 2000 Elsevier Science B.V. All rights reserved. Keywords: K-ras gene; Mutation; SSCP; Capillary electrophoresis; PCR
1. Introduction Lung cancer is one of the most prevalent cancers and the leading cause of cancer death in the world. The incidence of lung cancer is increasing dramatical*Corresponding author. Tel.: 186-371-6973-994. E-mail address: [email protected] (Z. Zhang). 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00310-7
206
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
ly in recent years. In order to find early biomarkers for lung cancer, it is necessary to study the mechanism of disease progression. Mutations in cellular ras gene have been strongly implicated in various stages of mammalian tumorigenesis. In human tumors, the point mutations which have been identified have largely been localized to codon 12,13 or 61 of three ras genes [1,2]: H-ras, K-ras and N-ras. Traditionally, mutations of these genes were detected by hybridization with radio-labelled DNA probes, allele-specific PCR and restriction fragment length polymorphism with polyacrylamide gel electrophoresis (PAGE). Many studies showed that gene mutations (such as base substitution, point mutation, deletion and insertion) could lead to a conformational change of single-stranded DNA, so the mobility shifts could be observed on the electrophoretogram [3,4]. However, the conventional method is laborious, timeconsuming and has low sensitivity. Capillary electrophoresis (CE) is rapid, automated, has high performance and reproducibility, and has recently been used to detect gene mutations. Single strand conformational polymorphism (SSCP) [5–7], restriction fragment length polymorphism (RFLP) [8,9], liagase chain reaction (LCR) [10], dideoxy fingerprinting amplification refractory mutation system (DFARM) [11], and chemical mismatch cleavage (CMC) [12] have been used to analyze gene mutations by CE. In the present work, SSCP was used with CE and laserinduced fluorescent (LIF) detector to detect the mutation of K-ras gene.
2. Materials and methods
2.1. Apparatus A P/ACE capillary electrophoresis instrument with laser-induced fluorescent detector ( lex 5 488 nm, lem 5 513 nm) was used. A fused silica capillary (37 cm 3 75 mm) was precoated by use of the method of Hjerten [13] with modifications.
2.2. Materials Acrylamide, N, N, N9, N9-tetramethylenediamine (TEMED) and ammonium peroxydisulfate (APS) were purchased from Sigma Chemical Co. 2-Amino-2(hydroxymethyl)-1,3-propanediol (Tris), boric acid, PCR Markers, Taq DNA polymerase, dimethyl sulfoxide (DMSO), blue phenol, xylene cyanole FF and formamide deionized were obtained from Sino-America Biotechnology Co. (SABC). dNTP was from Promega. Thiazole Orange (Fluorescent Molecular Probe) was supplied by Beckman. Primers were synthesized by CyberSyn Biotechnology Corporation (USA).
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
207
2.3. Preparation of sieving medium The short chain linear polyacrylamide gel (SLPA) was synthesized in our laboratory. The molecular mass and viscosity coefficient of SLPA were 2.563 10 5 and 1.25310 23 m 3 .kg 21 respectively. The different concentrations of SLPA were obtained by dissolving SLPA in running buffer (containing 100 mmol / l Tris 1100 mmol / l boric acid 12 mmol / l EDTA15% glycerol12 mg / l thiazole orange), using a magnetic stir bar and stir plate to stir the solution until SLPA was dissolved completely.
2.4. Amplification of K-ras gene by PCR DNA was isolated from lung tissues of rats, which included two groups (normal and tumor), by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook [14] with minor modifications. Primers used in the study were as the following: upstream primer: 59-ACA / TGT / TCT /AAT / ATA / GTC /AC-39, downstream primer: 59-AAA / GAA / TGG / TCC / TGC / ACC /AG-39. The PCR reactions were performed in a 50 ml buffer containing 50 pmol of each primer, 100 pmol of dATP, dCTP, dGTP, dTTP, 5 ml of 103PCR buffer, 0.5 mg template and 8% DMSO. Prior to PCR, the mixture was predenatured at 958C for 5 min and then 1.5 U DNA polymerase was added to the above solution. All PCR reactions were performed in a DNA cycle of SABC (Sino-America Biotechnology Company) and the reaction mixture was covered by 100 ml wax. The reaction mixture was subject to 30 cycles at 948C for 60 s, 648C for 50 s and 728C for 53 s, the extension time for the last cycle was 5 min at 728C, the length of DNA fragment generated by PCR was 212 bp.
2.5. SSCP by non-denatured PAGE Taking 10 ml of PCR products into equal volume of sample buffer containing 20 mmol / l EDTA, 96% formamide deionized, 0.05% bromphenol blue and 0.05% xylene cyanole FF, the above solution was heated at 978C for 5 min and then immediately chilled in ice water bath for 5 min; 10 ml of the above solution was applied to each lane of non-denatured polyacrylamide gel, which consisted of 5% polyacrylamide and 0.4% PEG. Electrophoresis was performed at 200 V for 4 h at 48C and the gel was stained with silver.
2.6. SSCP by capillary electrophoresis The inner surface of the capillary was coated by use of the method of Hjerten [13] with some modifications. Four percent short-chain linear polyacrylamide gel containing 100 mmol / l Tris plus 100 mmol / l boric acid, 2 mmol / l EDTA,
208
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
5% glycerol and 2 mg / l thiazole orange was filled into a capillary coated by a commercial capillary electrophoresis instrument (Beckman) and preconditioned in running buffer consisting of 100 mmol / l Tris, 100 mmol / l boric acid, 2 mmol / l EDTA, 5% glycerol and 2 mg / l thiazole orange for 10 min. After each run the capillary was purged with water for 5 min and again filled with 4% short linear chain polyacrylamide. Prior to electrophoresis, the sample was diluted 6-fold with sample buffer (20 mmol / l EDTA and 96% formamide deionized) and heated at 958C for 7 min and then immediately put into ice water bath for 5 min. After mixing of 0.1 ml thiazole orange with the above sample solution, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under the constant voltage of 8 kv. The DNA fragments were detected by LIF detector ( lex 5488 nm, lem 5513 nm) and the data were processed by Gold System Software.
3. Results In the study, the PAGE–SSCP results showed no mobility shift in the electrophoretogram for the 20 normal cases. A mobility shift did appear for samples 10 and 12 in the tumor group (16 cases). The migration pattern of K-ras gene from normal and tumor lung tissues is shown in Fig. 1. There were
Fig. 1. Electrophoretogram of SSCP for K-ras gene. N: Normal; M: Mutation. Tumor: lane 1–9; Control: lane 10.
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
209
Fig. 2. Electrophoretogram of SSCP by CE for K-ras gene. Upper: tumor (sample 10); Bottom: normal. N: normal; M: mutation.
abnormal band in the lane 1 (sample 10) and lane 3 (sample 12) in the electrophoretogram. This result demonstrated that K-ras gene of sample 10 and 12 mutated in the extron 1. The PCR products of normal and tumor sample were subject to SSCP by CE. The base resolution of two single strands of DNA fragments was achieved with short chain linear polyacrylamide at the concentration of 4%, samples 10 and 12 had 3 peaks in the electrophoretogram between 16.5 min and 18.5 min, and the result showed that samples 10 and 12 had one abnormal peak compared with the normal sample (Figs. 2, 3). Both results of PAGE–SSCP and CE–SSCP were identical.
4. Discussion Several water-soluble polymers, including cellulose derivatives, non-crosslinked linear polyacrylamide and poly (ethylene oxide) (PEO), have successfully been applied to the separation of dsDNA by CE [15]. However, cellulose derivatives and PEO were unsuitable for SSCP analysis due to inadequate resolution or high viscosity at the concentration required for adequate resolution. Short chain linear polyacrylamide has the advantage over long chain linear polyacrylamide due to its low viscosity and adequate resolution and could be filled into the capillary by use of a commercial capillary electrophoresis instrument.
210
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
Fig. 3. Electrophoretogram of SSCP by CE for K-ras gene. Upper: normal; Bottom: tumor (sample 12). N: normal; M: mutation.
Prior to SSCP analysis, the samples were heated to melt the double-stranded DNA molecules into a single strand. Because of the complementary nucleotide sequence of the two single strands of one DNA molecule, their folded conformation was different and this obviously led to different mobility in the non-denatured polyacrylamide gel. The amplification of K-ras gene by PCR generated a DNA fragment of 212 bp in the extron 1. Activation of K-ras oncogene is related to the pathogenesis of various human tumors. The incidence of ras gene mutation in the lung cancer is about 20%. Point mutations which lead to ras activation were confined to the codons 12, 13 and 61 of the gene. Point mutations in codon 12 are commonly seen. Both results of PAGE–SSCP and CE–SSCP demonstrated that K-ras gene of samples 10 and 12 mutated in the extron 1. One abnormal peak was observed respectively in electrophoretogram of PAGE and CE, so this result showed that only one type of mutation appeared for samples 10 and 12. Comparing CE–SSCP with PAGE–SSCP, CE–SSCP has higher resolution than PAGE–SSCE and the former method has the advantages of simplicity, rapidity, automation, high sensitivity and good suitability for clinical analysis.
Z. Zhang et al. / Clinica Chimica Acta 301 (2000) 205 – 211
211
References [1] Burmer GC, Rabinovitch PS, Loeb LA. Analysis of c-Ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA sequencing. Cancer Res 1989;49:2141–6. [2] DeFeo-Jones D, Scolnick EM, Koller R, Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature 1983;306:707–9. [3] Larsen LA, Andersen PS, Kanters JK, Jacobsen JR, Vuust J, Christiansen M. A single strand conformation polymorphism / heteroduplex method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome. Clin Chim Acta 1999;280:113–5. [4] Russeva MG, Janakiev PJ, Kirov SA et al. A simple method for detection of factor V R506Q (Leiden) mutation in dried blood spots. Clin Chim Acta 1999;284:89–92. [5] Kiyonori K, Keiko K, Wataru C, Sadao I, Moritoshi K. Fluorescence-based polymerase chain reaction–single-stranded conformation polymorphism analysis of p53 gene by capillary electrophoresis. J Chromatogr A 1996;744:311–20. [6] Jurgen G, Tanja W, Marion B, Markus N, Kurt O, Wolfgang H. Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis. J Chromatogr B 1999;724:239–47. [7] Arakawa H, Nakashiro S, Maedo M, Tsuji A. Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis. J Chromatogr A 1996;722:359–68. [8] Koay ES, Zhu M, Wehr T et al. Detection of apolipoprotein E genotype by capillary electrophoresis. Talanta 1998;45:673–81. [9] Gelfi C, Righetti P, Brancolini V, Cremonesi L, Ferrari M. Capillary electrophoresis in polymer networks for analysis of PCR products: detection of DF508 mutation in cystic fibrosis. Clin Chem 1994;40:1603–5. [10] Jing C, Mann AS, Keith RM, Larry JK, Peter W. Analysis of ligase chain reaction products amplified in a silicon glass chip using capillary electrophoresis. J Chromatogr A 1996;732:151–8. [11] Arakawa H, Vetanala K, Maeda M. Analysis of PCR by capillary electrophoresis and its application to the detection of single base substitution in genes. J Chromatogr A 1994;664:89–94. [12] Ren J, Ulvik A, Refsum H, Ueland PM. Chemical mismatch cleavage combined with capillary electrophoresis: detection of mutations in exon 8 of the cystathionine b-synthase gene. Clin Chem 1998;44:2108–14. [13] Hjerten S. High performance electrophoresis: elimination of electroendosmosis and solute adsorption. J Chromatogr 1985;347:191–8. [14] Sambrook J, Fitch EF, Manitatis T. In: A laboratory manual, New York: Cold Spring Harbor Laboratory Press, 1990, p. 467. [15] Righetti PG, Gelfi C. Non-isocratic capillary electrophoresis for detection of DNA point mutations. J Chromatogr B 1997;697:195–205.