US 10947589

US Patent 10947589 is titled "Varietal counting of nucleic acids for obtaining genomic copy number information."

Assignee: Cold Spring Harbor Laboratory
Inventors: James Hicks, Nicholas Navin, Jennifer Troge, Zihua Wang, Michael Wigler
Filing Date: March 7, 2016
Issue Date: March 16, 2021

Abstract:
A method for obtaining genomic copy number information unaffected by amplification distortion from genomic material is described. This method involves obtaining segments of genomic material, tagging these segments with substantially unique tags to create tagged nucleic acid molecules (each with one genomic segment and one tag), amplifying these tagged molecules using PCR, and then sequencing the PCR product to generate tag-associated sequence reads. Each tagged nucleic acid molecule is then assigned to a location on a genome by mapping the subsequence corresponding to the genomic segment. Finally, the number of tagged nucleic acid molecules with different tags assigned to the same genomic location is counted to determine the genomic copy number information.

Plain-language overview of each independent claim:

• Independent Claim 1: This claim describes a method for determining genomic copy number without the inaccuracies typically introduced by amplification. It involves taking fragments of genomic DNA, attaching unique "tags" to each fragment, amplifying these tagged fragments using PCR, and then sequencing them. By looking at the unique tags associated with each DNA sequence read, the method can count how many original DNA molecules were present at a specific location on the genome, thus providing accurate copy number information.

• Independent Claim 7: This claim focuses on determining mRNA copy number, again, without amplification distortion. It starts by converting mRNA transcripts into cDNA (either full-length or fragmented). These cDNA molecules (or fragments) are then tagged with unique identifiers, amplified via PCR, and sequenced. By counting the distinct tags mapped to a specific location on a cDNA library, the method provides an accurate measure of the original mRNA transcript abundance.

• Independent Claim 8: Similar to Claim 7, this claim also describes a method for obtaining mRNA copy number information, but with a specific tagging strategy. It involves generating "first order derivative strands" from mRNA using primers that bind to the polyA tail, then adding a polynucleotide tail to these strands. Subsequently, "second order derivative strands" are generated using primers that bind to this newly added tail. Crucially, the primers used in at least one of these polymerase reactions contain substantially unique tags, thereby generating the tagged nucleic acid molecules needed for amplification, sequencing, mapping to a cDNA library, and counting.

• Independent Claim 9: This claim details a method for obtaining DNA methylation information free from amplification distortion. It involves taking genomic DNA segments, adding a polynucleotide tail to their ends, and then performing a polymerase reaction with essentially unique primers that bind to this tail. This generates tagged nucleic acid molecules. These molecules are then separated into hemi-methylated and unmethylated groups. Each group is separately amplified, sequenced, and mapped to the genome. By counting the unique tagged molecules within each group at specific genomic locations, methylation information is obtained accurately.

• Independent Claim 10: This claim describes a "composition of matter" (essentially, a molecular product) derived from genomic material. This composition consists of tagged nucleic acid molecules created through a specific process. The process involves obtaining genomic segments, adding a polynucleotide tail, creating "first order derivative strands" using primers that bind to this tail, adding another polynucleotide tail, and then creating "second order derivative strands" using primers that bind to the second tail. The key is that at least one set of primers used in the derivative strand generation steps comprises substantially unique primers, thus incorporating the tags into the final product.

• Independent Claim 11: This claim also describes a composition of matter derived from genomic material, specifically tagged nucleic acid molecules. The process here involves obtaining genomic segments, adding a polynucleotide tail to form "zero-th order derivative strands." Then, primers are ligated to the 5' ends of these strands, followed by a polymerase reaction using different primers that bind to the polynucleotide tail. The polymerase must have 3'-5' proofreading activity. Similar to other claims, the primers in at least one of these steps incorporate substantially unique tags.

• Independent Claim 12: This claim outlines a composition of matter derived from mRNA transcripts, specifically tagged nucleic acid molecules. The process involves obtaining cDNA from mRNA (either full-length or fragmented) and then directly tagging this cDNA (or segments thereof) with substantially unique tags. Each resulting tagged nucleic acid molecule contains one cDNA (or segment) and one tag.

• Independent Claim 13: This claim describes a composition of matter derived from mRNA transcripts, similar to Claim 8's method, producing tagged nucleic acid molecules. The process includes obtaining mRNA transcripts, generating "first order derivative strands" using primers binding to the polyA tail, adding a polynucleotide tail to these, and then generating "second order derivative strands" using primers that bind to the added tail. The critical element is that the primers in at least one of the polymerase reaction steps include substantially unique primers, leading to the tagged nucleic acid molecules.

• Independent Claim 14: This claim is for a kit designed to determine nucleic acid copy number information without amplification distortion. The kit comprises a terminal transferase (an enzyme that adds nucleotides to the end of a DNA strand) and a plurality of substantially unique primers. These primers contain the unique tags and are specifically designed to hybridize to the polynucleotide tail added by the terminal transferase.

Regarding CAFC 2026 dockets for patent 10947589, I found no results. My search was for "CAFC 2026 dockets 10947589". I cannot definitively state there is no litigation without access to more extensive, real-time legal databases.