Pyrosequencing is a method used to sequence DNA by detecting the pyrophosphate (PPi) group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We report here the development of a method to solve this problem by using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety capping the 3-OH group to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the PPi for sequence determination, the 3-OH of the primer extension products is regenerated through different deprotection methods. Using an allyl or a 2-nitrobenzyl group as the reversible moiety to cap the 3-OH of the four nucleotides, we have synthesized two sets of 3-O-modified nucleotides, 3-O-allyl-dNTPs and 3-O-(2-nitrobenzyl)-dNTPs as reversible terminators for pyrosequencing. The capping moiety on the 3-OH of the DNA extension product is efficiently removed after PPi detection by either a chemical method or photolysis. To sequence DNA, templates containing homopolymeric regions are immobilized on Sepharose beads, and then extension-signal detection-deprotection cycles are conducted by using the nucleotide reversible terminators on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.nucleotide reversible terminator ͉ sequencing by synthesis D NA sequencing is a fundamental tool for biological science. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresisbased sequencing technology (2-5), a variety of new DNAsequencing methods have been investigated with an aim to eventually realize the goal of the $1,000 genome. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7-9), sequence-specific detection of DNA using engineered nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis approaches such as pyrosequencing (12), sequencing of single DNA molecules (13,14), and polymerase colonies (15) have been widely explored.Pyrosequencing is a method to sequence DNA by detecting the pyrophosphate (PPi) that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction (12). In this approach, each of the four nucleotides is added sequentially with a mixture of enzymes and substrates in addition to the usual polymerase reactio...