RESUMO
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.
Assuntos
DNA Polimerase Dirigida por DNA/química , Técnicas Genéticas , Nucleotídeos/química , Nucleotídeos/síntese química , Corantes/farmacologia , DNA/química , Primers do DNA/química , Modelos Químicos , Fosfatos/química , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Fatores de TempoRESUMO
Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.