Prior art

As a technical patent analyst, I have analyzed the prior art cited against US patent 12,264,358. The patent, titled "Method of selectively sequencing amplicons in a biological sample," focuses on immobilizing nucleic acids within their native biological context (e.g., a cell or tissue) inside a polymer matrix, then amplifying and sequencing them in situ to preserve their original three-dimensional spatial information. The key innovation appears to be the creation of a stable, cross-linked matrix of the amplicons themselves, allowing for numerous cycles of sequencing chemistry without degradation or loss of positional data.

The priority date for this patent is March 12, 2013. All references published before this date are considered prior art.

Below is an analysis of the most relevant prior art references and their potential to anticipate the claims of US 12,264,358 under 35 U.S.C. § 102.

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Analysis of Key Prior Art References

1. US 7,329,492 B2 (Mitra et al.)

• Full Citation: US Patent 7,329,492 B2, "In situ nucleic acid sequencing."

• Publication Date: February 12, 2008.

• Filing Date: September 2, 2004 (Priority: September 3, 2003).

• Brief Description: This patent is foundational for the method known as Fluorescent In Situ Sequencing (FISSEQ). It describes methods for determining the sequence of nucleic acids directly within a fixed and permeabilized biological specimen. The method involves amplifying the nucleic acids in situ to create localized colonies of DNA (amplicons), often within a polyacrylamide gel matrix to keep the amplicons from diffusing. The patent then describes performing sequencing-by-synthesis directly on these immobilized amplicons.

• Potential Anticipation under 35 U.S.C. § 102: This is a highly relevant reference that discloses many core elements of the '358 patent. It teaches:

  • Fixing and permeabilizing a biological sample.
  • Amplifying nucleic acids in situ to form localized amplicons. The use of a polyacrylamide gel matrix for this purpose is explicitly mentioned.
  • Sequencing the resulting amplicons in situ.

  A potential distinction, and the likely novel step in the '358 patent, is the specific teaching of incorporating functional moieties (e.g., aminoallyl dUTP) into the amplicons during amplification, and then performing a second cross-linking step to covalently link the amplicons to each other and/or the surrounding matrix. This post-amplification stabilization creates the "structurally and chemically stable" amplicon matrix emphasized in the '358 patent (see FIG. 8 description). If US 7,329,492 B2 only describes steric entrapment of amplicons within a gel rather than this specific covalent stabilization of the amplicons themselves, it may not fully anticipate claims requiring this step.

2. US 2008/0242560 A1 (Church et al.)

• Full Citation: US Patent Application Publication 2008/0242560 A1, "In situ analysis of nucleic acids."
• Publication Date: October 2, 2008.
• Filing Date: March 19, 2008 (Priority: March 21, 2007).
• Brief Description: This application, from the same inventors as the '358 patent, describes methods for in situ analysis of nucleic acids in cells and tissues. It discloses amplification techniques, including rolling circle amplification (RCA), performed directly within a fixed biological sample to generate localized signals that can be detected and sequenced. The primary goal is to obtain sequence information while preserving the spatial context of the nucleic acids.
• Potential Anticipation under 35 U.S.C. § 102: This reference strengthens the teachings of the in situ amplification and sequencing approach. It provides a clear motivation and method for preserving spatial information. However, like the Mitra '492 patent, it may not explicitly detail the formation of a covalently cross-linked amplicon matrix that is stabilized after amplification. The inventive step of the '358 patent appears to be this enhanced stabilization method, which allows the matrix to withstand harsh chemical treatments and over 50 cycles of sequencing. This reference is therefore more likely to be used in an obviousness rejection (35 U.S.C. § 103) rather than a direct anticipation.

3. US 8,435,741 B2 (Larsson et al.)

• Full Citation: US Patent 8,435,741 B2, "Padlock probes and their use."
• Publication Date: May 7, 2013 (with priority dates as early as 1997).
• Filing Date: December 11, 2008.
• Brief Description: This patent describes padlock probes, which are linear DNA probes that circularize upon binding to a specific target sequence. These circularized probes serve as ideal templates for rolling circle amplification (RCA), generating a highly localized and strong signal. The patent describes the use of this technique for in situ detection of nucleic acids.
• Potential Anticipation under 35 U.S.C. § 102: The '358 patent explicitly describes a workflow that involves reverse transcription, circularization of cDNA, and subsequent RCA—a process that relies on the principles taught by Larsson. This reference anticipates the in situ RCA step using circular templates. However, it does not teach the broader method of forming a stabilizing polymer matrix around these reactions within a cell, nor does it teach the subsequent cross-linking of the resulting RCA amplicons to form a durable matrix for sequencing. It anticipates a sub-step of the overall claimed process but not the entire combination.

4. US 7,709,198 B2 (Drmanac et al.)

• Full Citation: US Patent 7,709,198 B2, "Combinatorial probe anchor ligation methods for nucleic acid sequencing."
• Publication Date: May 4, 2010.
• Filing Date: December 21, 2006 (Priority: December 22, 2005).
• Brief Description: This patent relates to sequencing by ligation on arrays of amplified DNA "nanoballs." The method involves extracting DNA from a sample, amplifying it on a surface to create dense clusters, and then using combinatorial probes to read the sequence.
• Potential Anticipation under 35 U.S.C. § 102: This reference is unlikely to anticipate the claims of the '358 patent. The Drmanac method is fundamentally an ex situ process. It requires the extraction of nucleic acids from their native environment, thereby losing all spatial information, which is the central problem the '358 patent aims to solve. The '358 patent's background explicitly contrasts its in situ approach with such array-based methods.