ABSTRACT
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.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , Genetic Techniques , Nucleotides/chemistry , Nucleotides/chemical synthesis , Coloring Agents/pharmacology , DNA/chemistry , DNA Primers/chemistry , Models, Chemical , Phosphates/chemistry , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Time FactorsABSTRACT
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.
Subject(s)
Coloring Agents , DNA/chemistry , Deoxyribonucleotides/chemical synthesis , Dideoxynucleosides/chemical synthesis , Base Sequence , Energy Transfer , Indicators and ReagentsABSTRACT
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.
Subject(s)
DNA/chemistry , Fluorescence Resonance Energy Transfer/methods , Base Sequence , DNA-Directed DNA Polymerase , Fluorescent Dyes , Terminator Regions, GeneticABSTRACT
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.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Dideoxynucleosides/chemistry , Lysine/analogs & derivatives , Nucleotides/metabolism , Sequence Analysis, DNA/methods , Base Sequence , Chromatography, High Pressure Liquid/methods , Coloring Agents/chemistry , DNA/chemistry , DNA/genetics , Lysine/chemistry , Molecular Sequence Data , Molecular Structure , Nucleotides/chemistry , Nucleotides/genetics , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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.
