Energy Transfer Cassettes for Facile Labeling of Sequencing and PCR Primers

Berti, Lorenzo; Xie, Jin; Medintz, Igor L.; Glazer, Alexander N.; Mathies, Richard A.

doi:10.1006/abio.2001.5069

Cited by 36 publications

(43 citation statements)

References 21 publications

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“…The present study highlights the potential of alternative base labeling and pooling schemes to enhance 4 Straightforward modifications to the PRS coding/pooling scheme such that the forward and reverse extension reactions are performed simultaneously would reduce the number of reaction tubes by a factor of two. Another factor of two reduction is provided by the use of uniquely-labeled energy transfer (ET)-cassette primers (Berti et al 2001). For example, by pooling sample and reference templates amplified with uniquely-tailed PCR primers and performing PRS analysis using corresponding cassettelabeled primers, it is feasible to obtain forward and reverse PRS data on both templates with only four reaction tubes-the same number of tubes as dye-terminator sequencing.…”

Section: Discussionmentioning

confidence: 99%

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

Blazej

Paegel²,

Mathies³

2003

Genome Res.

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Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.Genetic studies are expanding past the identification of rare Mendelian diseases to encompass common complex disorders such as cancer, neurological diseases, autoimmune diseases, and cardiovascular diseases. The abundance and stability of single nucleotide polymorphisms (SNPs) has made them the DNA variation of choice for such endeavors. Due to the low penetrance of individual alleles, association studies designed to uncover significant genetic risk factors require the scoring of hundreds of thousands of SNPs (Kruglyak 1999) in several thousand samples (Dahlman et al. 2002). Multiplexed highthroughput genotyping methods are essential to accommodate this challenging number of SNPs and samples.Current methods of SNP detection include microarrays (Chee et al. 1996), allele-specific polymerase chain reaction (PCR; Liu et al. 1997), oligonucleotide ligation assays (Grossman et al. 1994;Samiotaki et al. 1994;Tobe et al. 1996), single-base extension (Syvanen et al. 1993;Pastinen et al. 1996;Shumaker et al. 1996), TaqMan (Livak et al. 1995, and molecular beacons (Tyagi et al. 1998). These methods are designed to interrogate SNPs in one sample at a time. Modifications such as kinetic PCR (Germer et al. 2000) and detection using denaturing high-performance liquid chromatography (HPLC; Giordano et al. 2001) can extend these procedures to multiplexed sample analysis. Each method offers a trade-off between sensitivity, throughput, parallel processing, user accessibility, and cost, and they all experience limitations due to local DNA hybridization characteristics. Selection of allelespecific probes is confined to the DNA sequence immediately adjacent to or surrounding the targeted polymorphism. This restriction can lead to nonuniform hybridization characteristics, ambiguous results, or the rejection of otherwise informative SNPs in a significant percentage of the markers. Additionally, novel polymorphisms are not detected by these approaches. These limitations are particularly important when studying functional or rare SNPs.Quality-based resequencing a...

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Section: Discussionmentioning

confidence: 99%

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

Blazej

Paegel²,

Mathies³

2003

Genome Res.

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“…The electrophoretic mobility shifts are minimal, and a 1.4-to 24-fold more intense signal is achieved compared to primers labeled with a single fluorophore. ET dyes have been successfully applied for multiplexing in DNA sequencing [142,146], SNP analysis [37], STR analysis [147], and PCR product sizing [148]. However, expensive, large, and sophisticated instrumentation is generally required for sensitive LIF detection with these dyes.…”

Section: Fluorescent Energy-transfer Dyesmentioning

confidence: 99%

DNA sequencing and genotyping in miniaturized electrophoresis systems

et al. 2004

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Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.

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“…ET-cassette-labeled primers were synthesized following Berti et al [8]. The synthesis began with the donor-spacer consisting of carboxyfluorescein (FAM) at the 3 0 -end, seven 1 0 ,2 0 -dideoxyribose (R) spacers, an amino-modified thymine (T) nucleotide, and monomethoxytrityl (MMT) protecting group at the 5 0 -end (Midland, Midland, TX).…”

Section: Et Cassette Synthesis and Primer Preparationmentioning

confidence: 99%

“…In this study, we explore the use of the universal ET cassette labeling method [8] for high-performance STR analyses at 16 commonly used forensic loci, including the 13 core or Combined DNA Index System (CODIS) loci required for the national database. A high-throughput microfabricated capillary array electrophoresis (mCAE) STR analysis system [9] is used for evaluating these reagents.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence energy transfer‐labeled primers for high‐performance forensic DNA profiling

et al. 2008

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A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.

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Energy Transfer Cassettes for Facile Labeling of Sequencing and PCR Primers

Cited by 36 publications

References 21 publications

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

DNA sequencing and genotyping in miniaturized electrophoresis systems

Fluorescence energy transfer‐labeled primers for high‐performance forensic DNA profiling

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