High-Throughput, High-Sensitivity Genetic Mutation Detection by Tandem Single-Strand Conformation Polymorphism/Heteroduplex Analysis Capillary Array Electrophoresis

Kourkine, Igor V.; Hestekin, Christa N.; Buchholz, Brett A.; Barron, Annelise E.

doi:10.1021/ac020025b

Cited by 62 publications

(96 citation statements)

References 29 publications

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“…With the increase of LPA concentrations, the resolution of ssDNA fragments was improved and all four peaks of the ssDNA conformers presented in the heterozygous sample were separated completely at 4% LPA. The small peaks clustered around the peaks of the ssDNA conformers probably represent primer-ssDNA constructs [24]. At 6% LPA, the resolution of ssDNA fragments was improved slightly but a longer electrophoresis time was required.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…However, tandem SSCP/HA through the snapcooling procedure produced similar results with the gradually cooling procedure. 19 The use of 10 mM Tris-HCl (PH 8.6) buffer for sample preparation provided high sensitivity and satisfactory results. Figure 4 shows the results of SSCP/HA of the P53 gene exon 7 of a mutant sample with homemade LPA.…”

Section: Establishment Of the Tandem Lif-ce-sscp/ha Methodsmentioning

confidence: 99%

Fluorescent-based Single-strand Conformation Polymorphism/Heteroduplex Capillary Electrophoretic Mutation Analysis of the P53 Gene

Zhao

Shi

et al. 2004

ANAL. SCI.

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Fluorescent-based single-strand conformation polymorphism (F-SSCP) analysis with capillary electrophoresis (CE) is the most common method for the detection of mutation because of its high sensitivity and resolution. In this study, we prepared an inexpensive linear polyacrylamide (LPA), and successfully applied it to CE-SSCP analysis and tandem CE-SSCP/heteroduplex analysis (HA) of the P53 gene on an ABI capillary genetic analyzer. A comparison of the sieving capabilities of a homemade LPA and commercial polydimethylacrylamide (PDMA) demonstrates that the homemade LPA has a higher resolution, a shorter analysis time, and is more suitable for tandem SSCP/HA than commercial PDMA. To show the usefulness, mutations of P53 gene exon 7 -8 in 37 tumor samples were investigated by using homemade LPA. The results indicate that 10 mutations were found in 9 of 37 cases; the majority of P53 mutations were missense mutations, and 70% were located in exon 7, which plays an important role in neoplastic progression in human tumorigenesis.

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“…90% [5]). By combining these two techniques (e.g., as in the capillary-based work of [6]) it has been reported that sensitivities of 100% can be achieved.…”

Section: General Aspectsmentioning

confidence: 99%

“…Significant strides have been made in developing microchip-based mutation detection using SSCP [6,20], HA [21,22] as well as restriction enzyme processing (e.g.,…”

Section: Microchip-based Mutation Detectionmentioning

confidence: 99%

An integrated method for mutation detection using on‐chip sample preparation, single‐stranded conformation polymorphism, and heteroduplex analysis

Vahedi¹,

Kaler²,

Backhouse³

2004

Electrophoresis

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This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.

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High-Throughput, High-Sensitivity Genetic Mutation Detection by Tandem Single-Strand Conformation Polymorphism/Heteroduplex Analysis Capillary Array Electrophoresis

Cited by 62 publications

References 29 publications

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

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

Fluorescent-based Single-strand Conformation Polymorphism/Heteroduplex Capillary Electrophoretic Mutation Analysis of the P53 Gene

An integrated method for mutation detection using on‐chip sample preparation, single‐stranded conformation polymorphism, and heteroduplex analysis

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