Synthesis of aminomethyl- and bis-aminomethyl-fluorescein energy transfer terminators.

Synthesis of aminomethyl and bis-aminomethylfluorescein derived energy transfer terminators is described.

Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases.

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.

Synthesis of novel piperidinyl linker based energy transfer terminators and their potential use in DNA sequencing.

Synthesis of novel piperidinyl linker based ET cassettes and terminators is described These novel terminators are evaluated in the DNA sequencing experiments using thermostable DNA polymerase.

Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays.

Nucleotides with a dye attached to the terminal phosphate with four or more phosphates (tetra- or pentaphosphates) are superior substrates than the corresponding triphosphates for DNA and RNA polymerases. When fluorogenic dyes are directly attached to the terminal phosphate, they can be released by the action of polymerase and alkaline phosphatase. The released dye changes color and fluorescence properties. The fluorescent signal can also be amplified by using multiple labeled nucleotides to detect small amounts of template. We have explored the utility of these nucleotides in a variety of applications including homogeneous SNP detection methods, DNA sequencing, and quantitation of PCR and RCA.

Synthesis of novel tyrosinyl FRET cassettes, terminators, and their potential use in DNA sequencing.

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.

Negatively charged, dye labeled-dideoxynucleotides for "direct-load" DNA sequencing.

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a "direct-load" DNA sequencing, obviating the laborious and time consuming post-reaction work-up.

Synthesis and energy transfer efficiency of FRET terminators derived from different linkers.

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5'-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.

Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

Novel cyanine dye-labeled dideoxynucleoside triphosphates for DNA sequencing.

Single color cyanine dye-labeled (Cy 5.0 and Cy 5.5) dideoxynucleoside-5'-triphosphates, or 'terminators', containing different spacer lengths were synthesized and evaluated for efficacy in DNA sequencing methods using a modified thermally stable DNA polymerase. The single color cyanine dye terminators were formulated into two separate sets of sequencing mixes, one for Cy 5.0 and the other for Cy 5.5, and evaluated on different automated sequencing platforms. Each set of mixes included two pyrimidine terminators with 17-atom linkers and two purine terminators with 10-atom linkers between the dye and the nucleotide. The two sets of cyanine dye-labeled terminators chosen for this cycle sequencing study produced improved band patterns with band uniformity similar to that obtained with dye-primer sequencing methods.

Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing.

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.

Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing.

Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.