High-performance genetic analysis using microfabricated capillary array electrophoresis microplates

Medintz, Igor L.; Paegel, Brian M.; Blazej, Robert G.; Emrich, Charles A.; Berti, Lorenzo; Scherer, James R.; Mathies, Richard A.

doi:10.1002/1522-2683(200110)22:18<3845::aid-elps3845>3.0.co;2-0

Cited by 89 publications

(55 citation statements)

References 101 publications

(128 reference statements)

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“…Homogeneous or solid-phase reaction formats are used in combination with fluorescent dyes or mass spectrometry (MS) for detection. Various high-density formats have been adapted to capillary electrophoresis (42 ), DNA microarrays (43 ), bead-based assays (44 ), and MS (45 ) to obtain cost-effective genotyping by miniaturization and a high degree of multiplexing. Recent publications have described ultrahigh-throughput approaches that enable the identification of up to hundreds of thousands (46 ) or millions (47 ) of genotypes within 24h.…”

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confidence: 99%

High-Level Multiplex Genotyping of Polymorphisms Involved in Folate or Homocysteine Metabolism by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

2004

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Background: Increased plasma total homocysteine (tHcy), a risk factor for cardiovascular disease, is related to genetic, environmental, and nutritional factors, in particular folate status. Future large epidemiologic studies of the genetic basis of hyperhomocysteinemia will require high-throughput assays for polymorphisms of genes related to folate and Hcy metabolism. Method: We developed a high-level multiplex genotyping method based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of 12 polymorphisms in 8 genes involved in folate or Hcy metabolism. The assay includes methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, cystathionine ␤-synthase (CBS) 844ins68 and 699C>T, transcobalamin II (TCII) 776C>G and 67A>G, reduced folate carrier-1 (RFC1) 80G>A, paraoxonase-1 (PON1) 575A>G and 163T>A, and betaine homocysteine methyltransferase (BHMT) 742G>A. Results: The failure rate of the assay was <1.7% and was attributable to unsuccessful DNA purification, nanoliter dispensing, and spectrum calibration. Most errors were related to identification of heterozygotes as homozygotes. The mean error rate was 0.26%, and error rates differed for the various single-nucleotide polymorphisms. Identification of CBS 844ins68 was carried out by a semiquantitative approach. The throughput of the

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confidence: 99%

High-Level Multiplex Genotyping of Polymorphisms Involved in Folate or Homocysteine Metabolism by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

2004

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“…System integration led to the 'lab-on-a-chip' concept with such multiple functions as PCR amplification, restriction digestion, separation, fraction collection, etc [10]. The progress in the field was summarized in several reviews in the past few years [9,[11][12][13][14][15][16][17][18][19][20][21].…”

Section: Genome Variabilitymentioning

confidence: 99%

“…To increase the speed of the technique, two oppositely orientated allele-specific primers (for the two allelic variants) can be utilized in a single-tube reaction. Allele-specific amplification in conjunction with microchip electrophoresis was reported by Medintz et al [11,32] for the analysis of three SNPs at three hereditary hemochromatosis loci in 96 individuals. The fluorescently labeled DNA samples were detected within the microchannels by a four-color rotary confocal fluorescence scanner.…”

Section: Dna Hybridization-based Techniquesmentioning

confidence: 99%

Genotyping with microfluidic devices

Szantai

Guttman

2006

Electrophoresis

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In the past few years, electrophoresis microchips have been increasingly utilized to interrogate genetic variations in the human and other genomes. Microfluidic devices can be readily applied to speed up existing genotyping protocols, in particular the ones that require electric field-mediated separations in conjunction with restriction fragment analysis, DNA sequencing, hybridization-based techniques, allele-specific amplification, heteroduplex analysis, just to list the most important ones. As a result of recent developments, microfabricated electrophoresis devices offer several advantages over conventional slab-gel electrophoresis, such as small sample volume requirement, low reagent consumption, the option of system integration and easy multiplexing. The analysis speed of microchip electrophoresis is significantly higher than that of any other electric field-mediated separation techniques. State-of-the-art microfluidic bioanalytical devices already claim their place in most molecular biology laboratories. This review summarizes the recent developments in microchip electrophoresis methods of nucleic acids, particularly for rapid genotyping, that will most likely play a significant role in the future of clinical diagnostics.

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“…The first successful efficient separation of dsDNA fragments on electrophoresis microchips was achieved in a cellulose derivative by Woolley [3]. Since then, microchip-based electrophoresis has made rapid advances in DNA analysis, such as PCR products [4 -6], double-stranded DNA fragments [3,7], and the sequencing of DNA [8,9], due to its high performance, speed, and resolution.…”

Section: Introductionmentioning

confidence: 99%

Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

Wang

Qin

Dai

et al. 2003

J of Separation Science

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Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix A novel protocol has been established to separate dsDNA fragments with high efficiency on glass chips by using an ultralow viscosity sieving matrix with added glucose. Low-molecular-weight hydroxypropylmethylcellulose (HPMC), with a viscosity nearly equivalent to that of water, was used to electrophoretically separate fluorescent intercalator-labeled double-stranded DNA (dsDNA) fragments on microfluidic glass chips. In comparison with conventional sieving protocols, low-molecular-weight HPMC as sieving matrix could result in reduced running cost and analysis time, in addition to a comparable separation efficiency of dsDNA fragments. In this paper, the addition of glucose was investigated to enhance the separation of DNA in the lowest viscosity polymer evaluated. The effect of staining dye and field strength were also evaluated. At an applied electric field strength of 200 V/cm, satisfactory resolution of the PBR322/HaeIII DNA marker could be achieved within 4 min by using 2% HPMC-5 with 6% glucose added. Coelectrophoresing PCR product along with bX174/HaeIII DNA sizing marker was also demonstrated by using the ultralow viscosity HPMC-5 solution on a glass chip.

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High-performance genetic analysis using microfabricated capillary array electrophoresis microplates

Cited by 89 publications

References 101 publications

High-Level Multiplex Genotyping of Polymorphisms Involved in Folate or Homocysteine Metabolism by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

High-Level Multiplex Genotyping of Polymorphisms Involved in Folate or Homocysteine Metabolism by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Genotyping with microfluidic devices

Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

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