Gene Expression Analysis by Means of Generating a Circularized Single Stranded Cdna Library

Simple SummaryContent extracted from patent full text and abstract with AI.

This invention describes a method and associated kits for gene expression analysis and nucleic acid profiling by generating circularized single-stranded complementary DNA (cDNA) libraries. The method involves converting nucleic acids (such as mRNA) into single-stranded cDNA with a blocked 3’ end, selectively degrading unused primers, removing the block, circularizing the cDNA, and then sequencing the resulting circular DNA without the need for prior amplification. The approach offers improved accuracy, flexibility in enzyme and nucleotide use, and higher proportions of meaningful sequencing reads compared to previous protocols.

Use CasesContent extracted from patent full text and abstract with AI.

• Gene expression profiling of cells or tissues in research and diagnostics.
• Single-cell RNA sequencing and transcriptomics studies.
• Clinical diagnostic tests for disease biomarkers based on gene expression.
• Monitoring of gene expression changes in response to treatments during drug discovery or therapy.
• Environmental or forensic sample analysis for the presence and activity of nucleic acids.
• Quality control or characterization of biological samples in biotechnology.

BenefitsContent extracted from patent full text and abstract with AI.

• High accuracy in quantifying gene expression, even at low sequencing depths.
• Enables production of sequencing-ready DNA libraries without the need for PCR amplification, reducing bias.
• Applicable to a wide variety of nucleic acids (mRNA, genomic DNA, etc.) and compatible with different enzymes and nucleotide chemistries.
• Higher yield of meaningful sequencing reads compared to previous methods, improving cost efficiency.
• Fragment lengths can be precisely controlled by adjusting ddNTP concentrations, enhancing versatility.
• Optimized for high-throughput workflows and scalable for multiplexed or pooled analyses.

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Patent Abstract

The present invention is directed to methods and kits for analysis of nucleic acid samples. The methods of the invention comprise the steps of a) providing nucleic acid; b) transcribing the nucleic acid to form single-stranded DNA having at its 3'-end a ddATP, ddCTP, ddGTP, or ddTTP nucleotide or a dATP, dCTP, dGTP, or dTTP nucleotide comprising a modified 3'-moiety by contacting the nucleic acid with a DNA polymerase, a primer and a mixture of (a) dNTPs and (b) at least one ddNTP or at least one dNTP comprising the modified 3'-moiety under conditions that allow the generation of the DNA, wherein the primer comprises a target-complementary region and optionally wherein the amounts of dNTPs are essentially equimolar; c) degrading the non-elongated primer; d) optionally purifying the DNA; e) contacting the DNA with a 3' to 5' exonuclease to remove the ddATP, ddCTP, ddGTP or ddTTP nucleotide or the dATP, dCTP, dGTP, or dTTP nucleotide comprising the modified 3'-moiety; f)i) contacting the DNA with a ssDNA ligase to circularize the DNA; or f)ii) contacting the DNA with (1) a linker molecule comprising a nucleic acid portion; and (2) a DNA ligase to ligate the DNA to the linker molecule; g) optionally amplifying the DNA; h) optionally quantifying the DNA by qPCR; and i) sequencing the DNA. The kits comprise the components necessary to perform the methods of the invention.