Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Abstract

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donoracceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm)

Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil

“…Both PCR tests were performed in duplicate, with two different samples to confi rm the diagnosis. The amplifi ed DNA fragment was directly sequenced in both directions using a Mega-BACE-sequencer (GE Healthcare; Little Chalfont, UK) (13) .The optimal alignment of the C-prM region using ClustalW (www.ncbi.nlm.nih.gov/pmc/articles/PMC308517) and MEGA version 5 (www.megasoftware.net) showed high identity among the nucleotide sequence of the sample (GenBank accession number 1735167) and other DENV-3 sequences deposited in GenBank. The paired nucleotide identity between our sample and other DENV-3 sequences ranged from 93.8% to 98.2% (14) .…”

Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

“…Recent studies also have demonstrated that an important route for identifying functional elements in the human genome is to sequence the genomes of many species that represent a wide sampling of the evolutionary tree (2). To overcome the limitations of the current sequencing technology based on electrophoresis using laser-induced fluorescence detection (3)(4)(5), a variety of new DNA-sequencing methods have been explored. Such techniques include pyrosequencing (6), MS sequencing (7-9), sequencing by hybridization (10), sequence-specific detection of singlestranded DNA using engineered nanopores (11), and sequencing of single DNA molecules (12) and polymerase colonies (13).…”

Toward a Complete Human Genome Sequence

“…Changes in DNA sequencing chemistry have led to substantial improvement in the quality and read length of the primary sequence, with no extra labor investment per sample. These changes include the introduction of fluorescent energy transfer dyes (Smith et al 1985;Ju et al 1995;Rosenblum et al 1997) for more sensitive labeling of DNA and engineered thermostable polymerases (''FY'') that are more processive and less discriminant in incorporating dideoxy nucleotides (Tabor and Richardson 1995). A complementary approach has been to increase the number of samples loaded per run.…”