US-12624351-B2 - Real-time detection of errors in oligonucleotide synthesis

Abstract

Fluorophores are used during the synthesis of oligonucleotides to achieve real-time quality control of the synthesis process. Fluorescence may indicate successful addition of individual nucleotides to a growing oligonucleotide strand or removal of a blocking group. The oligonucleotides may be created by enzymatic synthesis using terminal deoxynucleotidyl transferase (TdT). The synthesis is performed on an addressable array so that oligonucleotides with different sequences are created in parallel on different regions of the array. The oligonucleotide sequences are predetermined and the locations of synthesis on the array are controlled. Observed fluorescence is compared to expected locations of fluorescence as determined by the oligonucleotide sequences and the arrangement on the array. Thus, the fidelity of oligonucleotide synthesis is checked as synthesis proceeds. If a variation is found, a mitigating action is taken such as repeating addition of a species of nucleotide or repeating a deblocking step.

Inventors

• Bichlien Hoang NGUYEN
• Jake Smith
• Karin Strauss
• Robert Carlson

Assignees

• MICROSOFT TECHNOLOGY LICENSING, LLC

Dates

Publication Date

20260512

Application Date

20210519

Claims (19)

1. 1 . A method for real-time detection of errors in de novo synthesis of a plurality of oligonucleotides, the method comprising: (a) contacting an addressable array with a single species of nucleotide having a blocking group and a fluorophore; (b) capturing an image of the addressable array while exciting the fluorophore; (c) comparing the image to preexisting data indicating locations of expected fluorescence on the addressable array; (d) identifying an absence of fluorescence in at least one location on the image that corresponds to one of the locations of expected fluorescence on the addressable array; and (e) in response to identifying the absence of fluorescence, stopping synthesis at all array sites and discarding the plurality of oligonucleotides without completing the synthesis run.
2. 2 . The method of claim 1 , wherein the plurality of oligonucleotides are synthesized from four different species of nucleotide and a fluorophore associated with each species of nucleotide fluoresces a different color.
3. 3 . The method of claim 1 , wherein identifying the absence of fluorescence comprises determining that a number or frequency of spots on the addressable array with an absence of fluorescence exceeds a threshold.
4. 4 . A method for real-time detection of errors in de novo synthesis of a plurality of polymers, the method comprising: (a) incorporating physically detectable tags during synthesis of the plurality of polymers; (b) detecting the physically detectable tags at a plurality of locations on an addressable array, wherein detection of the physically detectable tags indicates formation of a covalent bond or non-covalent binding; (c) comparing the plurality of locations to expected locations, the expected locations are based on preexisting data specifying sequences of the plurality of polymers; (d) identifying a variation between the plurality of locations and the expected locations; and (e) in response to identifying the variation, stopping synthesis at all array sites and discarding the plurality of polymers without completing the synthesis run.
5. 5 . The method of claim 4 , wherein comparing the plurality of locations to expected locations comprises: comparing a captured representation of the addressable array showing individual locations of the plurality of locations to values generated for the individual locations of the plurality of locations from the preexisting data; and determining for each of the individual locations of the plurality of locations if the captured representation matches a corresponding value.
6. 6 . The method of claim 4 , wherein the addressable array is a microelectrode array with addressable electrodes and the discarding the plurality of polymers comprises activating a subset of the addressable electrodes at locations where there is the variation.
7. 7 . The method of claim 4 , further comprising saving data describing the variation in association with data describing the plurality of polymers, wherein the data describing the variation is further associated with an indication of a physical container in which the plurality of polymers are stored.
8. 8 . The method of claim 4 , further comprising saving data describing the variation in association with data describing the plurality of polymers, wherein the data describing the variation includes data indicating locations of deblocking and data indicating locations of polymer subunit addition, wherein the method further comprises calculating a synthesis yield for the plurality of polymers prior to discarding the plurality of polymers.
9. 9 . The method of claim 4 , wherein the physically detectable tags include fluorophores, the polymers are oligonucleotides, and the addressable array is a microelectrode array with addressable electrodes.
10. 10 . The method of claim 9 , wherein the de novo synthesis of a plurality of polymers comprises solid-phase, enzymatic oligonucleotide synthesis.
11. 11 . The method of claim 9 , wherein detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting fluorescence at a subset of the addressable electrodes, wherein the fluorescence indicates incorporation of a nucleotide.
12. 12 . The method of claim 9 , wherein the detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting a loss of fluorescence at a subset of the addressable electrodes, wherein the loss of fluorescence indicates removal of a blocking group.
13. 13 . The method of claim 9 , wherein the de novo synthesis of a plurality of polymers comprises templated enzymatic oligonucleotide synthesis and the fluorophores are incorporated into template strands, wherein detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting fluorescence at a subset of the addressable electrodes, and wherein fluorescence indicates removal of quenching blocking groups.
14. 14 . The method of claim 9 , wherein the de novo synthesis of a plurality of polymers comprises ligation of bit complexes.
15. 15 . The method of claim 14 , wherein a first bit complex encoding a first bit comprises a first fluorophore having a first color and quencher and a second bit complex encoding a second bit comprises a second fluorophore having a second color and a quencher, wherein detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting a change in fluorescence from the first color to the second color or from the second color to the first color at a subset of the addressable electrodes, wherein the change in fluorescence indicates hybridization of a bit complex.
16. 16 . The method of claim 14 , wherein the bit complexes include a fluorophore attached to a terminal phosphate, wherein detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting fluorescence, wherein fluorescence indicates hybridization of bit complexes to oligonucleotides.
17. 17 . The method of claim 14 , wherein the bit complexes include a fluorophore attached to a terminal phosphate, wherein detecting the physically detectable tags at a plurality of locations on an addressable array comprises detecting loss of fluorescence, wherein loss of fluorescence indicates ligation of the bit complexes to oligonucleotides.
18. 18 . The method of claim 4 , wherein the physically detectable tags comprise redox probes and detecting the physically detectable tags comprises voltammetry.
19. 19 . The method of claim 4 , wherein identifying the variation comprises determining that a number or frequency of variations exceeds a threshold.

Description

BACKGROUND Oligonucleotides may be synthesized for many reasons such as for use in basic scientific research, for medical applications, and even to encode digital data. There are many techniques for synthesizing oligonucleotides. One technique is enzymatic synthesis which uses a template-independent DNA polymerase such as terminal deoxynucleotidyl transferase (TdT). Any technique for oligonucleotide synthesis can introduce errors. If an error is introduced in de novo synthesis, the sequence of the oligonucleotide that is actually created is not the same as the intended sequence. Errors can include addition of an incorrect nucleotide or failure to add a nucleotide. Errors may cause experiments to fail, prevent a medical use from providing a benefit to a patient, or result in storage of incorrect digital data. Techniques for checking errors generally require sequencing of the oligonucleotides after synthesis is complete. Thus, synthesis errors are not detectable, if at all, until later in a process. If errors are systemic or widespread, an entire batch of oligonucleotides may be unusable. Continuing to create oligonucleotides when there is a systemic problem or high error rate may waste valuable reagents. For digital data storage applications, if the original data is not recorded elsewhere, errors in synthesis can result in a complete loss of data. Accordingly, it would be advantageous if there were a way to detect errors in real-time during synthesis of oligonucleotides. The following disclosure is made with respect to these and other considerations. SUMMARY This disclosure provides techniques that use physically detectable tags such as fluorophores to monitor steps in the synthesis of oligonucleotides or other polymers and identify variations that may indicate an error in synthesis. The oligonucleotides are synthesized using solid-phase techniques on the surface of an addressable array. The intended sequences of the oligonucleotides to be synthesized are known in advance. The addressable array controls the location of oligonucleotide growth so that oligonucleotides of different sequences may be synthesized on different portions of the same array. This technique can create a batch of oligonucleotides with different sequences. The addressable array may be implemented as a microelectrode array that has a large number of individually controllable electrodes. The sequences of the oligonucleotides and the locations where each oligonucleotide is created are used to determine where the physically detectable tags are expected in each round of synthesis. The specific event that results in detection of a physically detectable tag will vary with the type of tag and design of the synthesis system. In one implementation, nucleotides labeled with a fluorescent blocking group may be added to extend a growing oligonucleotide. Incorporation of the labeled nucleotides at specific locations on the addressable array can be detected by fluorescence. Deblocking before addition of another nucleotide can then be detected by a loss of fluorescence. The locations on the addressable array where the physically detectable tags are detected are compared to expected locations derived from the sequences of the oligonucleotides and the locations of synthesis. If there is a variation, such as not detecting fluorescence at a location on the addressable array where it was expected, this may trigger a mitigating action. The mitigating action may be abandonment of the synthesis run. Stopping further synthesis when there are errors can prevent wasting reagents. Alternatively, synthesis may be modified to correct or mitigate the error. For example, if expected fluorescence resulting from incorporation of nucleotides was not detected, the same species of nucleotide may be added again. Any variation from expected behavior may also be recorded in metadata that can be associated with the oligonucleotides once synthesis is complete. The metadata may include information such as the types and numbers of variations. This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter nor is it intended to be used to limit the scope of the claimed subject matter. The term “techniques,” for instance, may refer to system(s) and/or method(s) as permitted by the context described above and throughout the document. BRIEF DESCRIPTION OF THE DRAWINGS The Detailed Description is set forth with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different figures indicates similar or identical items. The figures are schematic representations and items shown in the figures are not necessarily to scale. FIG. 1 is a flow diagram showing