Microchip electrophoresis: a method for high-speed SNP detection

Schmalzing, Dieter; Belenky, Alexei; Novotny, M. A.; Koutny, Lance; Salas‐Solano, Oscar; El-Difrawy, Sameh; Adourian, Aram; Matsudaira, Paul; Ehrlich, D. J.

doi:10.1093/nar/28.9.e43

Cited by 79 publications

(46 citation statements)

References 17 publications

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“…The DGGE/CFPLA technique used in this study is quite sensitive, but it cannot readily be automated for routine application. In the context of the Human Genome Project, several novel methods for high-throughput identification of unknown point mutations, such as high-density oligonucleotide arrays, microchip capillary electrophoresis, MALDI-TOF mass spectrometry, or automated SSCP-based capillary electrophoresis have been established (12)(13)(14)(15)(16). Among these, hybridization to high-density oligonucleotide arrays has already successfully been applied to the identification of unknown heterozygous mutations even in large genes.…”

Section: Discussionmentioning

confidence: 99%

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Mutations at the Galactose-1-P-Uridyltransferase Gene in Infants with a Positive Galactosemia Newborn Screening Test

et al. 2002

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Newborn screening for galactosemia yields a high number of false-positive results. Confirmatory DNA testing for unknown galactosemia mutations on the initial positive sample using novel techniques of mutation detection tenders itself to reduce the recall rate. The potential benefits of confirmatory DNA testing, however, could be offset by the detection of a high percentage of galactosemia carriers, Duarte/galactosemia compound heterozygotes, and infants with benign sequence changes in the galactose-1-phosphate uridyltransferase (GALT) gene among infants with a positive biochemical screening test. Our aim was to determine the frequency and allelic distribution of all sequence changes in the GALT gene in 110 newborns with a positive total galactose screening test among 43,688 Austrian newborns screened consecutively. We found that only 20 of the 110 probands carried at least one known or novel candidate galactosemia mutation (one galactosemia homozygote, 7 Duarte/galactosemia compounds, 12 carriers) as judged by denaturing gradient gel electrophoresis and cleavage fragment length polymorphism analysis. Four novel galactosemia candidate mutations (Q9H, A46fsdelCAGCT, M129T, L342I) were identified. Sixty-seven probands had no detectable sequence changes and 23 carried only the benign Duarte or Los Angeles variant alleles or silent mutations. We conclude that a rapid and automatable confirmation test for unknown GALT mutations, e.g. on a high-density oligonucleotide array basis, has the potential to lower the recall rate of galactosemia screening in our population by about five-fold from 0.25 to 0.046%. Further research, however, will be required before the development of such a test can be advocated. Classical galactosemia (McKusick 230400) is an autosomal recessive inborn error of carbohydrate metabolism caused by a deficiency of GALT occurring with an incidence of 1:40,000 -60,000 in Caucasian populations (1). Newborn screening and dietary galactose restriction prevent the life-threatening neonatal symptoms of galactosemia. Despite dietary treatment, the majority of affected children develop long-term complications including mental retardation, speech deficits, and ovarian failure.Galactosemia screening by microbiological or biochemical tests for elevated blood galactose/galactose-1-P and/or reduced GALT activity yields a high rate of false positives (2). Only 1 in about 190 infants recalled in galactosemia screening programs across the United States will actually have galactosemia (3). Because of the potentially rapid and fatal course of the disease, newborns with positive test results have to be recalled urgently. This causes substantial anxiety among the families concerned (4) and increases the workload of the screening centers.DNA confirmatory testing for common mutations on the original dried blood spot with a positive initial biochemical screening result has been used to reduce the number of false positives in screening programs for cystic fibrosis (5) and hemoglobinopathies (6). Although two common mutation...

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Section: Discussionmentioning

confidence: 99%

“…Recently, however, several methods for highthroughput detection of unknown point mutations with high sensitivity and specificity have been developed (12)(13)(14)(15)(16). These novel techniques should allow the development of a confirmatory test for all sequence changes in the GALT gene.…”

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confidence: 99%

Mutations at the Galactose-1-P-Uridyltransferase Gene in Infants with a Positive Galactosemia Newborn Screening Test

et al. 2002

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“…Although sequencing is the most sensitive method for mutation detection, it is also the most expensive [1]. There have been great advances in the integration of microarray technologies (e.g., [2]) and in microchip-based enzymatic mismatch cleavage methods (e.g., [3]) but such methods have a significant falsepositive rate as well as a high cost per test [4]. Both SSCP and HA are mutation methods that are based on the self-assembly of DNA and are far less costly than sequencing although the sensitivity of each method is significantly lower (ca.…”

Section: General Aspectsmentioning

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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“…Conventional procedures involved the employment of mass spectrometry or gel electrophoresis for the discrimination of fragments produced by endonuclease cleavage. [3,4] These methods are time-consuming and of relatively high cost. Current approaches used to detect SNP include primarily the allele-specific DNA microarray, [5,6] the allele-specific TagMan assay, [7] the template-directed dye terminator incorporation assay [8] and the ligase detection reaction.…”

Section: Introductionmentioning

confidence: 99%

A Sensitive Fluorescence Anisotropy Method for Point Mutation Detection by Using Core–Shell Fluorescent Nanoparticles and High‐Fidelity DNA Ligase

Deng

Jiang

et al. 2007

Chemistry A European J

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The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core-shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core-shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.

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Microchip electrophoresis: a method for high-speed SNP detection

Cited by 79 publications

References 17 publications

Mutations at the Galactose-1-P-Uridyltransferase Gene in Infants with a Positive Galactosemia Newborn Screening Test

Mutations at the Galactose-1-P-Uridyltransferase Gene in Infants with a Positive Galactosemia Newborn Screening Test

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

A Sensitive Fluorescence Anisotropy Method for Point Mutation Detection by Using Core–Shell Fluorescent Nanoparticles and High‐Fidelity DNA Ligase

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