Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis

Arakawa, Hidetoshi; Nakashiro, Shuji; Maeda, Masako; Tsuji, Akio

doi:10.1016/0021-9673(95)00430-0

Cited by 46 publications

(31 citation statements)

References 13 publications

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“…However, for a routine mutation scanning, the requirements for slab gel electrophoresis and multiple running conditions are often very complicated and also time consuming. Capillary electrophoresis has recently been developed also for SSCP analysis [Arakawa et al, 1996] and shows high sensitivity when used in combination with automated DNA fragment mobility analysis [Inazuka et al, 1997]. The advantages of this method are high resolution, rapid analysis, and high reproducibility for DNA analysis.…”

Section: Introductionmentioning

confidence: 99%

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

et al. 2002

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Section: Introductionmentioning

confidence: 99%

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

et al. 2002

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“…This effect has been widely investigated [12,13,23,25]. Atha et al [25] systematically explored the effect of temperature on SSCP analysis of p53 mutations.…”

Section: Effect Of Running Temperaturementioning

confidence: 99%

“…The advantages of capillary electrophoresis instead of PAGE are speed, automation, resolution, and reproducibility. LIF detection has a 100-1000 times higher sensitivity than UV detection, and is widely applied in SSCP-CE [10][11][12][13][14][15]. Recently, published reports focused mostly on the effects of temperature, voltage, pH and DNA chain length on SSCP-CE performance [16,17].…”

Section: Introductionmentioning

confidence: 99%

“…To our knowledge, there are no reports on the systematic investigation of the effects of glycerol on SSCP-CE analysis though it is well-known that the addition of glycerol to sieving medium can enhance the resolution of single-strand DNA [10,11,14,15] and double-strand DNA [18,19]. SSCP in combination with LIF-CE has been successfully applied to the analysis of many human genes, such as p53 gene [10,11], ras oncogene [12], MTHFR gene [13], HFE gene [14], LDL receptor [15], and Vitamin D receptor gene [20]. In this paper, SSCP analysis of the hMLH1 gene by LIF-CE was for the first time developed.…”

Section: Introductionmentioning

confidence: 99%

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A single‐strand conformation polymorphism method by capillary electrophoresis with laser‐induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Shi

Zhao

et al. 2003

Electrophoresis

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A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples. IntroductionThe human hMLH1 gene plays a key role in the human DNA mismatch repair system, which recognizes and replaces mispaired nucleotides in DNA [1]. The inactivation of the gene results in increased genetic instability, which in turns leads to an increased rate of mutation in "gatekeeper" genes that regulate cell proliferation and death, and gives rise to cancer [2]. Searching for mutations of the hMLH1 gene has become more and more important in deciphering the genetic background of various cancers. Kuyper et al. [9]. The advantages of capillary electrophoresis instead of PAGE are speed, automation, resolution, and reproducibility. LIF detection has a 100-1000 times higher sensitivity than UV detection, and is widely applied in SSCP-CE [10][11][12][13][14][15]. Recently, published reports focused mostly on the effects of temperature, voltage, pH and DNA chain length on SSCP-CE performance [16,17]. To our knowledge, there are no reports on the systematic investigation of the effects of glycerol on SSCP-CE analysis though it is well-known that the addition of glycerol to sieving medium can enhance the resolution of single-strand DNA [10,11,14,15] and double-strand DNA [18,19].SSCP in combination with LIF-CE has been successfully applied to the analysis of many human genes, such as p53 gene [10,11]

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“…For the discrimination of single nucleotide difference, CE has been employed in a single-strand conformation polymorphism (SSCP) assay. 12,13 SSCP is based on the conformational change of a single-stranded DNA (ssDNA) containing single nucleotide substitution, resulting in an alteration of the electrophoretic mobility. The SSCP assay is regarded as being one of the simplest methods for detecting single nucleotide difference, and its application to microchip electrophoresis has also been achieved.…”

Section: Introductionmentioning

confidence: 99%

Capillary Electrophoretic Discrimination of Single Nucleotide Polymorphisms Using an Oligodeoxyribonucleotide-polyacrylamide Conjugate as a Pseudo-immobilized Affinity Ligand: Optimum Ligand Length Predicted by the Melting Temperature Values

2005

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We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg 2+ in the buffer solution.

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Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis

Cited by 46 publications

References 13 publications

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

A single‐strand conformation polymorphism method by capillary electrophoresis with laser‐induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Capillary Electrophoretic Discrimination of Single Nucleotide Polymorphisms Using an Oligodeoxyribonucleotide-polyacrylamide Conjugate as a Pseudo-immobilized Affinity Ligand: Optimum Ligand Length Predicted by the Melting Temperature Values

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