Methods And Compositions For Incorporating Nucleotides

What is this patent about?

’887 is related to the field of nucleic acid sequencing , specifically methods and compositions for determining the identity of nucleic acids in nucleotide sequences. This is achieved using data obtained from sequencing by synthesis (SBS) methods, a technique where specially designed nucleotides and DNA polymerases are used to read the sequence of chip-bound, single-stranded DNA templates. The background context involves the increasing amount of DNA sequence information and the need for high-fidelity sequencing methods.

The underlying idea behind ’887 is to improve the accuracy of sequencing by synthesis by addressing phenomena that can lead to errors in base calling. This is achieved through a combination of techniques, including correcting for sequence lead and lag , spectral crosstalk, and noise resulting from variations in illumination and/or filter responses. A key aspect is the use of a mixture of labeled and unlabeled reversible nucleotide analogue terminators, along with a cleaving agent and a cleaving agent scavenger.

The claims of ’887 focus on a method of incorporating labeled nucleotides into nucleic acid. This involves providing nucleic acid template molecules, a polymerase, a cleaving agent, and a mixture of labeled and non-labeled reversible nucleotide analogue terminators. The labeled terminators have a label that produces color and are labeled with at least one type of fluorescent dye. The method includes incorporating a first labeled nucleotide analogue, detecting the color of the fluorescent dye in two channels, removing the label and the 3'-OH capping group with the cleaving agent, and then incorporating a second nucleotide analogue.

In practice, the invention involves a cyclical process of nucleotide incorporation, detection, and cleavage. The use of both labeled and unlabeled nucleotides helps to drive the extension reaction to completion while minimizing the impact of the label on subsequent incorporations. The cleaving agent removes both the label and the 3'-OH blocking group, allowing the next nucleotide to be added. The cleaving agent scavenger is crucial for neutralizing any residual cleaving agent, preventing premature cleavage in the next cycle and reducing dephasing.

The invention differentiates itself from prior approaches by addressing specific error modes in sequencing by synthesis. Traditional methods may not adequately compensate for sequence lead and lag, spectral crosstalk, or the effects of residual cleaving agent. By incorporating a cleaving agent scavenger and using a mixture of labeled and unlabeled nucleotides, the invention provides a more robust and accurate sequencing method, leading to longer read lengths and improved base calling fidelity. The use of specific mathematical models and matrix operations further enhances the accuracy of the sequencing process.