Methods and ion sensitive field effect transistor arrays for monitoring nucleic acid sequencing-by-synthesis reactions.

Abstract

The present invention provides methods and app. relating to field effect transistor (FET) arrays including large FET arrays for monitoring chem. and/or biol. reactions such as nucleic acid sequencing-by-synthesis reactions. More generally, arrays including large arrays of chemFETs may be employed to detect and measure static and/ or dynamic amts. or concns. of a variety of analytes (e.g., hydrogen ions, other ions, nonionic mols. or compds., etc.) in a variety of chem. and/or biol. processes (e.g., biol. or chem. reactions, cell or tissue cultures or monitoring, neural activity, nucleic acid sequencing, etc.) in which valuable information may be obtained based on such analyte measurements. Accordingly, the systems and methods shown herein provide uses for chemFET arrays that involve detection of analytes in soln. and/ or detection of change in charge bound to the chemFET surface. Methods are presented for maintaining or increasing signal (and thus signal-to-noise ratio) when using very large chemFET arrays, and in particular when increasing the d. of a chemFET array (and concomitantly decreasing the area of any single chemFET within the array). It has been found that as chemFET area decreases in order to accommodate an ever increasing no. of sensors on a given array, the signal that can be obtained from a single chemFET may in some instances decrease. The invention provides in some aspects and embodiments methods for overcoming this limitation. Of particular importance is the ability to increase signal during a nucleic acid synthesis reaction, and more particularly increasing signal attributable to hydrogen ions that are generated during such a reaction. In this context, some methods of the invention involve increasing the efficiency with which released (or generated) hydrogen ions are detected. It has been detd. in the course of our work that released hydrogen ions may be sequestered in a reaction chamber that overlays the chemFET, thereby precluding their detection by the chemFET. This disclosure therefore provides in some aspects methods and compns. for reducing buffering capacity of the soln. within which such reactions are carried out or reducing buffering capacity of solid supports that are in contact with such soln. In this way, a greater proportion of the hydrogen ions released during a nucleic acid synthesis reaction (such as one that is part of a sequencing-by-synthesis process) are detected by the chemFET rather than being for example sequestered by buffering components in the reaction soln. or chamber. [on SciFinder(R)]