Gene expression analysis

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

This patent discloses methods and kits for gene expression analysis that enable unbiased, amplification-free sequencing of nucleic acids (especially mRNA) by generating single-stranded DNA, selectively cleaving it at points of uracil incorporation, circularizing the DNA, and then sequencing these circles. The approach uniquely incorporates dUTP during DNA synthesis, then uses a combination of uracil deglycosylase and an endonuclease to generate cleavage sites for efficient DNA processing, allowing direct high-throughput sequencing without PCR amplification and its associated biases.

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

• Gene expression profiling in research, including transcriptome analysis of single cells or cell populations.
• Clinical diagnostics such as cancer, infectious diseases, or genetic disorders where unbiased RNA quantification from patient samples is beneficial.
• Pharmaceutical drug discovery and biomarker validation by quantifying gene activity in various biological samples.
• Biotechnology companies developing sequencing products or workflows for high-throughput genetic analyses.
• Forensic analysis and genetic identification where accurate nucleic acid quantitation is important.
• Agricultural genomics for plant and animal breeding studies requiring reliable gene expression data.

BenefitsContent extracted from patent full text and abstract with AI.

• Eliminates the need for PCR amplification, reducing bias and enabling more accurate quantification of rare transcripts.
• Allows for high-throughput, automatable, and cost-effective gene expression analyses.
• Enables unbiased detection of low-abundance nucleic acid molecules, improving sensitivity in research and diagnostics.
• Works directly from a range of biological samples, including single cells, cell populations, and diverse tissue types.
• Provides a simplified workflow suitable for next-generation sequencing platforms.
• Can be multiplexed by use of barcoded primers, increasing throughput and scalability for large studies.

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Inventors & Applicants

Patent Abstract

The present invention is directed to methods and kits for gene analysis. The methods of the invention comprise the steps of providing nucleic acid; synthesis of a single-stranded DNA that is complementary to said nucleic acid molecule by contacting the nucleic acid with a DNA polymerase, a primer and a mixture of dNTPs under conditions that allow the generation of the DNA, wherein the primer comprises a target-complementary region and wherein the dNTP mixture comprises dATP, dGTP, dCTP, dTTP and dUTP; cleaving the DNA 5' to dU sites by (i) contacting the DNA with an uracil deglycosylase to generate abasic sites at positions of dUTP incorporation in the DNA; and (ii) contacting the DNA with an apurinic/apyrimidinic (AP) endonuclease; contacting the DNA with a ssDNA ligase to circularize the DNA; and sequencing the circularized cDNA. The kits comprise the components necessary to perform the methods of the invention.